Sample records for mice flow cytometric

  1. Clonal evolution demonstrated by flow cytometric DNA analysis of a human colonic carcinoma grown in nude mice

    Vindeløv, L L; Spang-Thomsen, M; Visfeldt, J


    A spontaneous change in DNA content of a human colonic carcinoma grown in nude mice was observed fortuitously. The tumor initially had a G1 cell DNA content of 1.3 times that of normal cells. Flow cytometric DNA analysis showed in transplant generation 56 the appearance of a new subpopulation whi...... evolution of a tumor would be less pronounced if old subpopulations often become extinct as new ones emerge. Heterogeneity of human tumors is of clinical importance because the individual subpopulations may have different sensitivity patterns to antineoplastic drugs....

  2. Effect of 17 beta-oestradiol on growth curves and flow cytometric DNA distribution of two human breast carcinomas grown in nude mice

    Brünner, N; Spang-Thomsen, M; Vindeløv, L


    The effect of 17 beta-oestradiol on a "receptor positive" and on a "receptor negative" human breast carcinoma grown in nude mice was studied. Experimental growth data were used to determine the effect on tumour growth. Flow cytometric DNA analysis (FCM) performed on tumour tissue obtained...

  3. Flow cytometric gating for spleen monocyte and DC subsets: differences in autoimmune NOD mice and with acute inflammation.

    Dong, Matthew B; Rahman, M Jubayer; Tarbell, Kristin V


    The role of antigen presenting cells (APCs) in the pathogenesis of autoimmune and other inflammatory diseases is now better understood due to advances in multicolor flow cytometry, gene expression analysis of APC populations, and functional correlation of mouse to human APC populations. A simple but informative nomenclature of conventional and plasmacytoid dendritic cell subsets (cDC1, cDC2, pDC) and monocyte-derived populations incorporates these advances, but accurate subset identification is critical. Ambiguous gating schemes and alterations of cell surface markers in inflammatory condition can make comparing results between studies difficult. Both acute inflammation, such as TLR-ligand stimulation, and chronic inflammation as found in mouse models of autoimmunity can alter DC subset gating. Here, we address these issues using in vivo CpG stimulation as an example of acute inflammation and the non-obese diabetic (NOD) mouse as a model of chronic inflammation.We provide a flow cytometric antibody panel and gating scheme that differentiate 2 monocytic and 3DC subsets in the spleen both at steady state and after CpG stimulation. Using this method, we observed differences in the composition of NOD DCs that have been previously reported, and newly identified increases in the number of NOD monocyte-derived DCs. Finally, we established a protocol for DC phosphoflow to measure the phosphorylation state of intracellular proteins, and use it to confirm functional differences in the identified subsets. Therefore, we present optimized methods for distinguishing monocytic and DC populations with and without inflammation and/or autoimmunity associated with NOD mice.

  4. Flow cytometric analysis of the graft-versus-Leukemia-effect after hematopoietic stem cell transplantation in mice.

    Schmidt, Felix; Hilger, Nadja; Oelkrug, Christoper; Svanidze, Ellen; Ruschpler, Peter; Eichler, Wolfram; Boldt, Andreas; Emmrich, Frank; Fricke, Stephan


    Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model.

  5. Radio-protective effect of vitamin E on spermatogenesis in mice exposed to γ-irradiation: a flow cytometric study

    C.Songthaveesin; J.Saikhun; Y.Kitiyanant; K.Pavasuthipaisit


    Aim: To investigate the effect of vitamin E on the radioprotection of spermatogenesis and chromatin condensation of spermatozoa during passage through the epididymis in mice exposed to irradiation. Methods: Adult outbred male ICR mice were orally administered natural vitamin E (VE,D-a-tocopheryl acetate) at 400 IU/kg for 7 days before exposure to 1 Gy of y-irradiation. The animals were sacrificed at day 1,7,14,21, 28, 35 and 70 post-irradiation (IR) and the percentage of testicular germ cells and epididymal sperm chromatin condensation was analyzed using flow cytometry. Results: Serum D-a-tocopheryl acetate levels were 47.4±3.2μg/dL in the treatedgroup, yet it could not be detected in the control group. The testicular weight of irradiated mice pretreated with VE+IR was significantly (P<0.05) higher than that of those without VE treatment (IR) at day 14 and 21 post-irradiation. The percentage of primary spermatocytes (4C) in the VE+IR group was comparable to the controls but significantly (P<0.05) higher than those in the IR group from day 7 to 35 post-irradiation. The percentage of round spermatids (1C) in the VE+IR group was also significantly (P<0.05) higher than those in the IR group at day 28 postirradiation. The primary spermatocytes:spermatogonia ratio in the IR group was significantly (P<0.05) declined at day 7 to 35 post-irradiation when compared to the VE+IR and control groups. The round spermatid:spermatogonia ratio in the VE+IR group was significantly (P<0.05) higher than that of the IR group at day 14 and 28 post-irradiation.The chromatin condensation of epididymal spermatozoa measured by propidium iodide uptake was not affected by 1 Gy of γ-irradiation. Conclusion: The administration of VE prior to irradiation protects spermatogenic cells fromradiation. (Asian J Androl 2004 Dec; 6: 331-336)

  6. Recent advances in flow cytometric cell sorting.

    Osborne, Geoffrey W


    The classification and separation of one cell type or particle from others is a fundamental task in many areas of science. Numerous techniques are available to perform this task; however, electrostatic cell sorting has gained eminence over others because, when combined with the analysis capabilities of flow cytometry it provides flexible separations based on multiple parameters. Unlike competing technologies, such as gradient or magnetic separations that offer much larger total throughput, flow cytometric cell sorting permits selections based on various levels of fluorescent reporters, rather the complete presence or absence of the reporter. As such, this technology has found application in a huge range of fields. This chapter aims to describe the utility of single-cell sorting with particular emphasis given to index sorting. This is followed by two recently developed novel techniques of sorting cells or particles. The first of these is positional sorting which is useful in cell-based studies where sorting can proceed and produce meaningful results without being inherently dependant on prior knowledge of where gates should be set. Secondly, reflective plate sorting is introduced which positionally links multiwell sample and collection plates in a convenient assay format so that cells in the collection plate "reflect" those in the sample plate.

  7. Howard University Flow Cytometric Sorter For Research and Education


    SECURITY CLASSIFICATION OF: Howard University’s newly acquired Fluorescence Activated Cytometric Sorter (FACS) has been integrated into the new flow...Research Administrative Services Washington, DC 20059 -0001 11-Mar-2015 ABSTRACT Number of Papers published in peer-reviewed journals : Number of Papers...published in non peer-reviewed journals : Final Report: Howard University Flow Cytometric Sorter For Research and Education Report Title Howard

  8. Flow cytometric enumeration of marine viral populations at low abundances

    Mojica, K.D.A.; Evans, C.; Brussaard, C.P.D.


    Flow cytometric enumeration has advanced our ability to analyze aquatic virus samples and thereby our understanding of the ecological role viruses play in the oceans. However, low virus abundances are underestimated using the current flow cytometry (FCM) protocol. Our results revealed that low

  9. Flow cytometric immunoassay for sulfonamides in raw milk

    Keizer, de W.; Bienenmann-Ploum, M.; Bergwerff, A.A.; Haasnoot, W.


    Sulfonamide antibiotics are applied in veterinary medicine for the treatment of microbial infections. For the detection of residues of sulfonamides in milk, a multi-sulfonamide flow cytometric immunoassay (FCI) was developed using the Luminex MultiAnalyte Profiling (xMAP) technology. In this automat

  10. Technical discussions II - Flow cytometric analysis

    Cunningham, A; Cid, A; Buma, AGJ


    In this paper the potencial of flow cytometry as applied to the aquatic life sciences is discussed. The use of flow cytometry for studying the ecotoxicology of phytoplankton was introduced. On the other hand, the new flow cytometer EUROPA was presented. This is a multilaser machine which has been sp

  11. Technical discussions II - Flow cytometric analysis

    Cunningham, A; Cid, A; Buma, AGJ


    In this paper the potencial of flow cytometry as applied to the aquatic life sciences is discussed. The use of flow cytometry for studying the ecotoxicology of phytoplankton was introduced. On the other hand, the new flow cytometer EUROPA was presented. This is a multilaser machine which has been sp

  12. Flow cytometric detection of aberrant chromosomes

    Gray, J.W.; Lucas, J.; Yu, L.C.; Langlois, R.


    This report describes the quantification of chromosomal aberrations by flow cytometry. Both homogeneously and heterogeneously occurring chromosome aberrations were studied. Homogeneously occurring aberrations were noted in chromosomes isolated from human colon carcinoma (LoVo) cells, stained with Hoechst 33258 and chromomycin A3 and analyzed using dual beam flow cytometry. The resulting bivariate flow karyotype showed a homogeneously occurring marker chromosome of intermediate size. Heterogeneously occurring aberrations were quantified by slit-scan flow cytometry in chromosomes isolated from control and irradiated Chinese hamster cells and stained with propidium iodide. Heterogeneously occurring dicentric chromosomes were detected by their shapes (two centrometers). The frequencies of such chromosomes estimated by slit-scan flow cytometry correlated well with the frequencies determined by visual microscopy.

  13. Flow cytometric studies of human osteosarcoma.

    Mankin, H J; Gebhardt, M C; Springfield, D S; Litwak, G J; Kusazaki, K; Rosenberg, A E


    A number of recent studies have emphasized the potential value of flow cytometry as a "marker" to assess the malignity and therefore to help predict the biologic behavior of neoplasms, including bone tumors. Using propidium iodide and a home-built flow cytometer, the authors have studied the DNA distribution in 95 patients with osteosarcoma and determined the percentage of cells in diploidy, S-phase, tetraploidy, and aneuploidy. Using these values and a derived one, mean DNA concentration, it was possible to demonstrate the extent of the abnormalities observed in this group of neoplasms and show their severity as compared with the normal pattern. When the data are compared against disease-free survival and total survival, correlations were noted that, although weak, suggested that some patterns were predictive of increased risk of metastasis and death. The effect of treatment could also be assessed by evaluating the pattern before and after chemotherapy and correlating these with survival. It seems likely that with some improvement in technology, flow cytometry will be of value in the future in assessing the prognosis for osteosarcoma and predicting whether treatment has been effective.

  14. Flow cytometric detection method for DNA samples

    Nasarabadi,Shanavaz (Livermore, CA); Langlois, Richard G. (Livermore, CA); Venkateswaran, Kodumudi S. (Round Rock, TX)


    Disclosed herein are two methods for rapid multiplex analysis to determine the presence and identity of target DNA sequences within a DNA sample. Both methods use reporting DNA sequences, e.g., modified conventional Taqman.RTM. probes, to combine multiplex PCR amplification with microsphere-based hybridization using flow cytometry means of detection. Real-time PCR detection can also be incorporated. The first method uses a cyanine dye, such as, Cy3.TM., as the reporter linked to the 5' end of a reporting DNA sequence. The second method positions a reporter dye, e.g., FAM.TM. on the 3' end of the reporting DNA sequence and a quencher dye, e.g., TAMRA.TM., on the 5' end.

  15. Dose-dependent effect of 17 beta-estradiol determined by growth curves and flow cytometric DNA analysis of a human breast carcinoma (T61) grown in nude mice

    Brünner, N; Spang-Thomsen, M; Vindeløv, L


    of the treatment was evaluated using growth curves and flow cytometric DNA analysis. The treatment induced a dose-dependent growth delay and dose-dependent changes in the cell cycle distribution. The cell cycle changes comprised a decrease in the G1 phase, an accumulation of cells in the S phase, and an increasing...

  16. Flow-cytometric Analysis of Bacillus anthracis Spores

    D. V. Kamboj


    Full Text Available Flow-cytometric technique has been established as a powerful tool for detection andidentification of microbiological agents. Unambiguous and rapid detection of Bacillus anthracisspores has been reported using immunoglobulin G-fluorescein isothiocyanate conjugate againstlive spores. In addition to the high sensitivity, the present technique could differentiate betweenspores of closely related species, eg, Bacillus cereus and Bacillus subtilis using fluorescenceintensity. The technique can be used for detection of live as well as inactivated spores makingit more congenial for screening of suspected samples of bioterrorism.

  17. Automated High-Dimensional Flow Cytometric Data Analysis

    Pyne, Saumyadipta; Hu, Xinli; Wang, Kui; Rossin, Elizabeth; Lin, Tsung-I.; Maier, Lisa; Baecher-Allan, Clare; McLachlan, Geoffrey; Tamayo, Pablo; Hafler, David; de Jager, Philip; Mesirov, Jill

    Flow cytometry is widely used for single cell interrogation of surface and intracellular protein expression by measuring fluorescence intensity of fluorophore-conjugated reagents. We focus on the recently developed procedure of Pyne et al. (2009, Proceedings of the National Academy of Sciences USA 106, 8519-8524) for automated high- dimensional flow cytometric analysis called FLAME (FLow analysis with Automated Multivariate Estimation). It introduced novel finite mixture models of heavy-tailed and asymmetric distributions to identify and model cell populations in a flow cytometric sample. This approach robustly addresses the complexities of flow data without the need for transformation or projection to lower dimensions. It also addresses the critical task of matching cell populations across samples that enables downstream analysis. It thus facilitates application of flow cytometry to new biological and clinical problems. To facilitate pipelining with standard bioinformatic applications such as high-dimensional visualization, subject classification or outcome prediction, FLAME has been incorporated with the GenePattern package of the Broad Institute. Thereby analysis of flow data can be approached similarly as other genomic platforms. We also consider some new work that proposes a rigorous and robust solution to the registration problem by a multi-level approach that allows us to model and register cell populations simultaneously across a cohort of high-dimensional flow samples. This new approach is called JCM (Joint Clustering and Matching). It enables direct and rigorous comparisons across different time points or phenotypes in a complex biological study as well as for classification of new patient samples in a more clinical setting.

  18. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET

    Shin-Rong Lee


    Full Text Available Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs. Förster resonance energy transfer (FRET-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC, and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.

  19. An automated flow cytometric micronucleus assay for human lymphocytes.

    Schreiber, G A; Beisker, W; Braselmann, H; Bauchinger, M; Bögl, K W; Nüsse, M


    A new flow cytometric method is presented for scoring micronuclei (MN) in human lymphocytes after in vitro gamma-irradiation. Fifty to fifty-five hours after PHA-stimulation, the frequency of micronuclei per nucleus and the fraction of cells in the second cell cycle were measured using flow cytometry. All data were automatically analysed using our DAS-software package. Eight individual linear-quadratic dose response curves derived from five donors revealed inter- and intra-individual variabilities of all curve parameters. Since also an age dependence was found for spontaneous MN-frequencies and for the linear curve parameter, a combined linear-quadratic age-dose-effect model was used to fit the data. The 90% prediction intervals show that a reliable individual dose estimation for donors aged between 23 and 54 years cannot be achieved for exposures below 1 Gy.

  20. Comparative flow cytometric analysis of immunofunctionalized nanowire and nanoparticle signatures.

    Prina-Mello, Adriele; Whelan, Aine M; Atzberger, Ann; McCarthy, Joseph E; Byrne, Fiona; Davies, Gemma-Louise; Coey, J M D; Volkov, Yuri; Gun'ko, Yurii K


    Flow cytometry is one of the gold-standard techniques used in clinical medicine for quantitative immunoassaying. The continuous development of its probes, commonly fluorescent nanoparticles, is important. Lately, the introduction of quantitative multiplexed immunoassay has challenged the use of nanoparticles as probes. Functionalized fluorescent silica-based magnetic nanowires are investigated under flow cytometry as a novel probe category. The preparation and full characterization of these multimodal nanowires is reported and compared to those of silica-based magnetic nanoparticles by flow cytometry. Full characterization includes transmission electron microscopy and fluorescence microscopy imaging, flow cytometric assaying, superconducting quantum interference device (SQUID) magnetization, and Mössbauer spectroscopy measurements. This work shows that loaded silica nanowires have intrinsic geometrical advantages when compared to similar spherical particles due to their unique "flow cytometry fingerprint" when utilized as magnetic carriers for immunodetection applications. These advantages account for a 17% yield in detecting the functional binding between THP-1 and ICAM-1, by utilizing a much lower concentration than that required for the nanoparticles.

  1. Flow cytometric detection of anti-gliadin antibodies.

    Presani, G; Perticarari, S; Mangiarotti, M A


    A very sensitive solid-phase fluorescent immunoassay to detect anti-alpha-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 microns diameter, coated with alpha-gliadin. Serum-specific antibodies bound to the alpha-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques (r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

  2. A flow cytometric in vivo chalone assay using retransplanted old murine JB-1 ascites tumour cells.

    Barfod, N M


    A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G1 DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.

  3. Flow cytometric detection of immunoglobulin light chain in hematolymphoid immunophenotyping

    Xu Dongsheng


    During B cell development and maturation, the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH)and light chain genes,rearrange to associate one of a number of variable, diverse, and joining gene segments. A single mature Bcell expresses an IgH chain and either a kappa or lambda light chain, which is known as allelic or isotypic exclusion. In normal or reactive conditions, lymphoid cells comprise the mixtures of lymphocytes with either kappa or lambda expression. The norreal proportion of kappa to lambda (κ/λ ratio) is within the range of 0. 5 - 3. 0 in peripheral blood or bone marrow and 1.2 2.7 in lymph nodes[1].The most useful feature for diagnosing mature B cell neoplasm is light chain restriction or monotypic staining with κ or λ light chain. Currently, it is a common assumption that demonstration of light chain restriction in a B lymphocyte population is generally considered proof of monoclonality and indicates malignancy although monotypic B cell populations have been infrequently demonstrated in patients with no definitive evidence ofB cell malignancy[2-4]. The most common flow cytometric analysis for determining B cell monotype is the percent κ and λimmunoglobulin light chains.Because of the importance of light chain restriction in the diagnosis of B cell neoplasm, anti immunoglobulin antibodies (e. g. anti-κ and anti-λ) are vital tools in the detection of monotypic B cell populations. Accurate determination of surface light chain expression depends on many factors, such as proper washing procedure, lysing solution, type of antibody used, specimen type or lymphoma type[5]. This article will discuss some common problems encountered in flow cytometric(FCM)deterruination of surface immunoglobulin light chain expression inhematolymphoid immunophenotyping.%@@ During B-cell development and maturation,the antigen receptor,which is encoded by the immunoglobulin heavy-(IgH) and light-chain genes,rearrange to associate one of a number of

  4. Flow cytometric and laser scanning microscopic approaches in epigenetics research.

    Szekvolgyi, Lorant; Imre, Laszlo; Minh, Doan Xuan Quang; Hegedus, Eva; Bacso, Zsolt; Szabo, Gabor


    Our understanding of epigenetics has been transformed in recent years by the advance of technological possibilities based primarily on a powerful tool, chromatin immunoprecipitation (ChIP). However, in many cases, the detection of epigenetic changes requires methods providing a high-throughput (HTP) platform. Cytometry has opened a novel approach for the quantitative measurement of molecules, including PCR products, anchored to appropriately addressed microbeads (Pataki et al. 2005. Cytometry 68, 45-52). Here we show selected examples for the utility of two different cytometry-based platforms of epigenetic analysis: ChIP-on-beads, a flow-cytometric test of local histone modifications (Szekvolgyi et al. 2006. Cytometry 69, 1086-1091), and the laser scanning cytometry-based measurement of global epigenetic modifications that might help predict clinical behavior in different pathological conditions. We anticipate that such alternative tools may shortly become indispensable in clinical practice, translating the systematic screening of epigenetic tags from basic research into routine diagnostics of HTP demand.

  5. Flow cytometric immunophenotypic characteristics of plasma cell leukemia

    Barbara Kruk


    Full Text Available The aim of this prospective study was to define the flow cytometric characteristics of simultaneously investigated bone marrow and peripheral blood plasma cells antigens expression in 36 plasma cell leukemia (PCL patients. The immunophenotypic profile of plasma cells was determined with a panel of monoclonal antibodies. The antigen expression intensity was calculated as relative fluorescence intensity (RFI. Bone marrow plasma cells showed expression of particular antigens in the following proportion of cases: CD49d 100%, CD29 94%, CD54 93%, CD44 83%, CD56 60%, CD18 26%, CD11b 29%, CD11a 19%, CD117 27%, CD71 30%, CD126 100% and CD19 0%, while the expression of those antigens on peripheral blood plasma cells was present in the following percentage of patients: CD49d 100%, CD29 96%, CD54 93%, CD44 95%, CD56 56%, CD18 50%, CD11b 53%, CD11a 29%, CD117 26%, CD71 28%, CD126 100% and CD19 0%. The expression of CD54 was significantly higher than that of adhesion molecules belonging to the integrin b2 family: CD11a, CD18 and CD11b, on both bone marrow and peripheral blood cells (p < 0.01. Expression of CD18, CD11a and CD11b was differential between two cell compartments: lower on bone marrow and higher on peripheral blood cells. We found that plasma cells in the bone marrow of patients with plasma cell leukaemia showed significantly greater granularity and size than those in the peripheral blood (p = 0.0001 and p = 0.04, respectively. However, no differences in cell size or granularity were revealed between bone marrow plasma cells from patients with PCL and multiple myeloma. In conclusion, impaired expression of adhesion molecules such as CD11a/CD18 (LFA-1 or CD56 may explain hematogenic dissemination characterizing PCL. The following pattern of adhesion molecule expression according to the proportion of plasma cells expressing a given antigen in peripheral blood and bone marrow and arranged in diminishing order may be established: CD49d > CD44 > CD54

  6. Cell kinetics in a model of artificial skin. An immunohistochemical and flow cytometric analysis

    A Casasco


    Full Text Available Bioengineered organs raised in vitro are candidate substitutes for natural organs in biological, pharmacological and clinical applications. We have studied cell kinetics in a human skin equivalent (HSE using a combined immunohistochemical and flow cytometric approach. Morphological analysis has shown that, relative to unstimulated natural skin, cell proliferation mainly occurs in the basal layer of the epidermal equivalent. Immunohistochemical and flow cytometric measurements of the growth fraction suggested a cell turnover comparable to that of natural skin. Immunohistochemical labelling indices matched well with flow cytometric data. These observations are consistent with morphological and histochemical data demonstrating normal cell differentiation and tissue architecture in HSE and suggest that such HSE may be a usefull substitute for human skin.

  7. Cluster Analysis of Flow Cytometric List Mode Data on a Personal Computer

    Bakker Schut, Tom C.; Bakker schut, T.C.; de Grooth, B.G.; Greve, Jan


    A cluster analysis algorithm, dedicated to analysis of flow cytometric data is described. The algorithm is written in Pascal and implemented on an MS-DOS personal computer. It uses k-means, initialized with a large number of seed points, followed by a modified nearest neighbor technique to reduce

  8. Long-term storage of samples for flow cytometric DNA analysis

    Vindeløv, L L; Christensen, I J; Keiding, N


    A simple procedure for long-term storage of cells for flow cytometric DNA analysis was developed and tested. The cells were stored as single cells or fine-needle aspirates suspended in a citrate buffer with dimethylsulfoxide (DMSO), or as small blocks of tissue from solid tumors. The cells were...

  9. Flow cytometric sorting of paraffin-embedded tumor tissues considerably improves molecular genetic analysis

    Jordanova, ES; Corver, WE; Vonk, MJ; Leers, MPG; Riemersma, SA; Schuuring, E; Kluin, PM


    The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric s

  10. Flow cytometric determination of circulating immune complexes with the indirect granulocyte phagocytosis test

    Terstappen, L.W.M.M.; Grooth, de B.G.; Nolten, G.M.J.; Napel, ten C.H.H.; Berkel, van W.; Greve, J.


    A method for the determination of circulating immune complexes (CIC) was adapted for flow cytometric analysis. Human granulocytes were used to phagocytose IgG-bearing CIC of serum from systemic lupus erythematosus (SLE) patients. A method for labeling the phagocytosed CIC with FITC-conjugated anti-h

  11. Discrimination of bromodeoxyuridine labelled and unlabelled mitotic cells in flow cytometric bromodeoxyuridine/DNA analysis

    Jensen, P O; Larsen, J K; Christensen, I J


    Bromodeoxyuridine (BrdUrd) labelled and unlabelled mitotic cells, respectively, can be discriminated from interphase cells using a new method, based on immunocytochemical staining of BrdUrd and flow cytometric four-parameter analysis of DNA content, BrdUrd incorporation, and forward and orthogona...

  12. A flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk

    Holm, C.; Mathiasen, T.; Jespersen, Lene


    AIMS: The present study describes a flow cytometric technique for quantification and differentiation of bacteria in bulk tank milk according to the main cause of elevated counts. METHODS AND RESULTS: A total of 75 Danish bulk tank milk samples exceeding the grading level of 3.0 x 10(4) CFU ml(-1)...

  13. A flow-cytometric method to evaluate eosinophil-mediated uptake of probiotic Lactobacillus reuteri.

    Kraemer, Laura S; Brenner, Todd A; Krumholz, Julia O; Rosenberg, Helene F


    Eosinophils are resident leukocytes of gut mucosa. Here we present a combined flow cytometric-antibiotic protection assay to identify mouse eosinophils capable of bacterial uptake, specifically, Gram-positive Lactobacillus reuteri, in studies performed ex vivo. The assay may be adapted for use in vivo.

  14. Simultaneous preparation of RNA and nuclei for Northern blot and flow cytometric analysis

    Tesfaigzi, J.; Jaramillo, R. [Inhalation Toxicology Research Inst., Albuquerque, NM (United States)


    Several methods have been developed to quantify RNA synthesis during the progression of the cell cycle. The rate of RNA synthesis can be detected during different stages of the cell cycle by staining cells with agents that intercalate with nucleic acids. For example, following staining of mammalian cells with acridin orange, the green and red fluorescence that correlates with DNA and RNA content, respectively, can be analyzed by flow cytometry. Increase in RNA content during the progression of cells through the cell cycle can be measured after staining with acridin orange. RNA synthesis resulting from the stimulation of quiescent cells with various growth factors has also been demonstrated by labeling cells with bromo-uridine and using the anti-bromo-deoxyuridine antibody. These methods allow measurement of the overall RNA content in cells; however, they do not allow the measurement of the levels of specific mRNAs throughout the cell cycle. Current methods to quantify specific mRNAs generally require the preparation of a large number of cells (5--10 {times} 10{sup 6} cells) to carry out flow cytometric analyses and to isolate RNA for Northern blot analysis or solution hybridization. In this report, the authors describe a method of simultaneously preparing RNA and nuclei for Northern blot and flow cytometric analyses, respectively. The minimum number of nuclei required to obtain flow cytometric data and the effect of conserving nuclei in methanol for several days are also presented.

  15. Results of external quality control study in flow cytometric acute lymphoblastic leukemia diagnostics

    A. M. Popov


    Full Text Available Comparison of interpretation of acute lymphoblastic leukemia (ALL flow cytometric diagnostics data was the aim of the study. Immunophenotyping data obtained from 10 patients with ALL were analysed separately in 26 laboratories from Russian Federation and Kazahstan. Results comparison showed four main type of discordance: B-lineage ALL diagnostics during heavy bone marrow regeneration, great variability of T-ALL interpretation, complexity of ambiguous lineage acute leukemia and, finally, very different report types, unique for each laboratory. All these problems are the serious obstacles for standardization of flow cytometric ALL diagnostics in multicenter setting. Continuation of similar QC rounds following by consecutive discussions with further development of consensus diagnostic algorithm could be the first step for standardization of ALL immunophenotyping in Russian Federation and CIS countries.

  16. Flow cytometric assessment of viability of lactic acid bacteria

    Bunthof, C.J.; Bloemen, K.; Breeuwer, P.; Rombouts, F.M.; Abee, T.


    The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA b

  17. Flow cytometric DNA ploidy analysis of ovarian granulosa cell tumors

    D. Chadha; C.J. Cornelisse; A. Schabert (A.)


    textabstractAbstract The nuclear DNA content of 50 ovarian tumors initially diagnosed as granulosa cell tumors was measured by flow cytometry using paraffin-embedded archival material. The follow-up period of the patients ranged from 4 months to 19 years. Thirty-eight tumors were diploid or near-dip

  18. Detachment and flow cytometric quantification of seagrass-associated bacteria.

    Trevathan-Tackett, Stacey; Macreadie, Peter; Ralph, Peter; Seymour, Justin


    A new protocol was developed to detach bacteria from seagrass tissue and subsequently enumerate cells using flow cytometry (FCM). A method involving addition of the surfactant Tween 80 and vortexing resulted in maximum detachment efficiency of seagrass attached bacteria, providing a robust protocol for precisely enumerating seagrass-associated bacteria with FCM. Using this approach we detected cell concentrations between 2.0×10(5) and 8.0×10(6)cells mg(-1) DW tissue.

  19. Strategies for immunophenotyping and purifying classical Hodgkin lymphoma cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Fromm, Jonathan R; Wood, Brent L


    Flow cytometry is an established technique to immunophenotype hematopoietic neoplasms. While the diagnosis of classical Hodgkin lymphoma (CHL) has commonly been made using paraffin sections, we have recently demonstrated that the neoplastic Hodgkin and Reed-Sternberg (HRS) cells of CHL can be identified by flow cytometry. Using 6- and 9-color flow cytometric assays, CHL can be immunophenotyped with 85-90% sensitivity and nearly 100% specificity. Analysis of this data requires using established gating strategies to help in the identification of putative HRS cell populations. Interestingly, HRS cells bind to reactive T cells (HRS-T cell rosetting) and this phenomenon can be identified and utilized diagnostically by flow cytometry. In addition, the reactive T cells of CHL show characteristic immunophenotypic changes by flow cytometry and these changes can suggest a diagnosis of CHL. Finally, these principles can be employed to rapidly purify HRS cells using flow cytometric cell sorting. This manuscript provides experimental protocols for immunophenotyping CHL by flow cytometry as well as purifying the HRS cells via flow cytometric cell sorting.

  20. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    Crissman, Harry A.; Cui, H. H. (H. Helen); Steinkamp, J. A.


    Most flow cytometry (FCM) applications monitor fluorescence intensity to quantitate the various cellular parameters; however, the fluorescence emission also contains information relative to the fluorescence lifetime. Recent developments in FCM (Pinsky et al., 1993; Steinkamp & Crissman, 1993; Steinkamp et al., 1993), provide for the measurement of fluorescence lifetime which is also commonly referred to as fluorescence decay, or the time interval in which a fluorochrome remains in the excited state. Many unbound fluorochromes have characteristic lifetime values that are determined by their molecular structure; however, when the probe becomes bound, the lifetime value is influenced by a number of factors that affect the probe interaction with a target molecule. Monitoring the changes in the lifetime of the probe yields information relating to the molecular conformation, the functional state or activity of the molecular target. In addition, the lifetime values can be used as signatures to resolve the emissions of multiple fluorochrome labels with overlapping emission spectra that cannot be resolved by conventional FCM methodology. Such strategies can increase the number of fluorochrome combinations used in a flow cytometer with a single excitation source. Our studies demonstrate various applications of lifetime measurements for the analysis of the binding of different fluorochromes to DNA in single cells. Data presented in this session will show the utility of lifetime measurements for monitoring changes in chromatin structure associated with cell cycle progression, cellular differentiation, or DNA damage, such as induced during apoptosis. Several studies show that dyes with specificity for nucleic acids display different lifetime values when bound to DNA or to dsRNA. The Phase Sensitive Flow Cytometer is a multiparameter instrument, capable of performing lifetime measurements in conjunction with all the conventional FCM measurements. Future modifications of this

  1. Flow cytometric data analysis of circulating progenitor cell stability.

    Mahar, Ernestine A; Mou, Liping; Hayek, Salim S; Quyyumi, Arshed A; Waller, Edmund K


    A recent publication by Mekonnen et al. demonstrated that among women with non-obstructive coronary artery disease, higher levels of circulating progenitor cells in the blood (CPC), were associated with impaired coronary flow reserve [1]. We performed a quality control assessment of the stability of circulating blood progenitor cells in blood samples stored at 4 °C, to determine the time period during which blood samples can be analyzed and yield consistent data for progenitor cell content. Healthy volunteers (n=6) were recruited and underwent phlebotomy, and blood was stored in EDTA tubes at 4 °C. Flow cytometry was performed to quantitate progenitor cell subsets at 0-4 h, 24 h, and 48 h post phlebotomy. All processed samples were fixed with 1% Paraformaldehyde and 1,000,000 total data events were collected. We found no significant differences in PC data for both CD34+ (P=0.68 for one-way ANOVA) and CD34+/CD133+ (P=0.74 for one-way ANOVA).

  2. Flow cytometric data analysis of circulating progenitor cell stability

    Ernestine A. Mahar


    We performed a quality control assessment of the stability of circulating blood progenitor cells in blood samples stored at 4 °C, to determine the time period during which blood samples can be analyzed and yield consistent data for progenitor cell content. Healthy volunteers (n=6 were recruited and underwent phlebotomy, and blood was stored in EDTA tubes at 4 °C. Flow cytometry was performed to quantitate progenitor cell subsets at 0–4 h, 24 h, and 48 h post phlebotomy. All processed samples were fixed with 1% Paraformaldehyde and 1,000,000 total data events were collected. We found no significant differences in PC data for both CD34+ (P=0.68 for one-way ANOVA and CD34+/CD133+ (P=0.74 for one-way ANOVA.

  3. DNA flow cytometric analysis in variable types of hydropic placentas

    Fatemeh Atabaki pasdar


    Full Text Available Background: Differential diagnosis between complete hydatidiform mole, partial hydatidiform mole and hydropic abortion, known as hydropic placentas is still a challenge for pathologists but it is very important for patient management. Objective: We analyzed the nuclear DNA content of various types of hydropic placentas by flowcytometry. Materials and Methods: DNA ploidy analysis was performed in 20 non-molar (hydropic and non-hydropic spontaneous abortions and 20 molar (complete and partial moles, formalin-fixed, paraffin-embedded tissue samples by flow cytometry. The criteria for selection were based on the histopathologic diagnosis. Results: Of 10 cases histologically diagnosed as complete hydatiform mole, 9 cases yielded diploid histograms, and 1 case was tetraploid. Of 10 partial hydatidiform moles, 8 were triploid and 2 were diploid. All of 20 cases diagnosed as spontaneous abortions (hydropic and non-hydropic yielded diploid histograms. Conclusion: These findings signify the importance of the combined use of conventional histology and ploidy analysis in the differential diagnosis of complete hydatidiform mole, partial hydatidiform mole and hydropic abortion.

  4. Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae

    Solomon, J.A.; Hand, R.E. Jr.; Mann, R.C.


    Lipid accumulation in three species of microalgae was investigated with flow cytometry (FCM) and transmission electron microscopy (TEM). Previous studies using batch cultures of a algae have led to the assumption that lipid accumulation in microalgae is a gradual process requiring at least several days for completion. However, FCM reveals, through changes in the chlorophyll:lipid ratio, that the time span required for individual cells to change metabolic state is short. Simultaneous FCM measurements of chlorophyll and nile red (neutral lipid) fluorescence in individual cells of nitrogen-deficient Isochrysis populations revealed a bimodal population distribution as one stage in the lipid accumulation process. The fact that two discrete populations exist, with few cells in an intermediate stage, suggests rapid response to a liqid trigger. Interpretations of light and electron microscopic observations are consistent with this hypothesis. The time required for an entire population to achieve maximum lipid content is considerably longer than that required for a single cell, due to the variation in response time among cells. In this study high lipid cultures were sometimes obtained by using FCM to separate high lipid cells from the remainder of the population. FCM holds much promise for strain enhancement but considerable developmental work, directed at providing more consistent results, remains to be done. 8 refs., 35 figs.

  5. Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug-induced cell cycle perturbations in vitro

    Engelholm, S A; Spang-Thomsen, M; Vindeløv, L L


    A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small-cell carcinoma of the lung. Three tumors with different...... sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow...... cytometric DNA analysis. Melphalan produced a dose-related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan...

  6. IVBT-documented platelet function correlates with flow cytometric data.

    Hoffmann, J; Bonacker, G; Kretschmer, V; Schulzki, T; Heimanns, J


    Thrombocytopenic patients with identical platelet counts often show different bleeding tendencies owing to significant differences in the platelet function. This could be demonstrated by the in vitro bleeding test (IVBT). Using flow cytometry, we tried to find characteristics of platelet antigen expression in order to explain these differences in function. Thirty patients with bone marrow hypoplasia receiving 65 platelet transfusions (mainly from a cell separator) were observed for 3 to 29 days. Size, granulation and fluorescence of platelet-rich plasma (n = 522 samples) were evaluated using monoclonal antibodies against GP IIIb (collagen receptor), GP IIb/IIIa (fibrinogen receptor) and GP Ib (thrombin receptor). We defined separate gates for each antibody using the results from 50 normals and by laying an orthograde cross over the gate to divide the gate into four equal quadrants. The platelet populations were divided into four different groups according to the occlusion time (OT) of the IVBT and the Simplate time (ST). The thrombocytes with the most impaired function (OT > or = 485 s/ST > 30 min) had significantly less platelet fluorescence when marked with antibodies against GP IIIb and GP Ib than those with short OT and ST (OT platelet fluorescence when marked with anti-GP IIIb and anti-GP Ib than thrombocytopenic patients, who had a spontaneous platelet rise beyond 30,000 platelets/microliters a few days later. One day after platelet transfusion, significantly more platelets with high GP IIIb and Ib expression could be found. We were also able to document better transfusion efficacy of platelet concentrates with high GP IIIb and Ib expression. Finally, patients with high bleeding scores showed less GP Ib expression on the platelets than patients with low bleeding scores. In summary, the IVBT-documented platelet function clearly corresponded to an increased expression of the collagen receptor and the thrombin receptor of platelets.

  7. Karyological and flow cytometric evidence of triploid specimens in Bufo viridis (Amphibia Anura

    D Cavallo


    Full Text Available Karyological and flow cytometric (FCM analyses were performed on a group of 14 green toads of the Bufo viridis species from seven Eurasian populations. Both approaches gave concordant results concerning the DNA ploidy level. All the populations examined were represented exclusively by diploid or tetraploid specimens, except one, where triploids were found. Results evidenced an interpopulation variability in DNA content against the same ploidy level, as well as an unusually high number of triploids in a particular reproductive place. The origin of polyploidy and the presence and persistence of a high number of triploids in a particular population are discussed.

  8. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

    Nielsen, Morten Hjuler; Beck-Nielsen, Henning; Andersen, Morten Nørgaard;


    BACKGROUND AND AIM: Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold...... of detection of a new generation BD FACSAria™ III digital flow cytometer. METHODS: We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (.... The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS) exposing MPs binding Lactadherin was determined. RESULTS: By using a flow cytometric approach we...

  9. Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria.

    Barbesti, S; Citterio, S; Labra, M; Baroni, M D; Neri, M G; Sgorbati, S


    Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology. Copyright 2000 Wiley-Liss, Inc.

  10. Flow cytometric methods to investigate culture heterogeneities for plant metabolic engineering.

    Gaurav, Vishal; Kolewe, Martin E; Roberts, Susan C


    Plant cell cultures provide an important method for production and supply of a variety of natural products, where conditions can be easily controlled, manipulated, and optimized. Development and optimization of plant cell culture processes require both bioprocess engineering and metabolic engineering approaches. Cultures are generally highly heterogeneous, with significant variability amongst cells in terms of growth, metabolism, and productivity of key metabolites. Taxus cultures produce the important anti-cancer agent Taxol((R)) (i.e., paclitaxel) and have demonstrated significant variability amongst cell populations in culture with regard to paclitaxel accumulation, cell cycle participation, and protein synthesis. To fully understand the link between cellular metabolism and culture behavior and to enable targeted metabolic engineering approaches, cultures need to be studied at a single cell level. This chapter describes the application of plant cell flow cytometric techniques to investigate culture heterogeneity at the single cell level, in order to optimize culture performance through targeted metabolic engineering. Flow cytometric analytical methods are described to study Taxus single cells, protoplasts, and nuclei suspensions with respect to secondary metabolite accumulation, DNA content, cell size, and complexity. Reproducible methods to isolate these single particle suspensions from aggregated Taxus cultures are discussed. Methods to stain both fixed and live cells for a variety of biological markers are provided to enable characterization of cell phenotypes. Fluorescence-activated cell sorting (FACS) methods are also presented to facilitate isolation of certain plant cell culture populations for both analysis and propagation of superior cell lines for use in bioprocesses.

  11. Flow cytometric analysis of microbial contamination in food industry technological lines – initial study

    Katarzyna Czaczyk


    Full Text Available Background. Flow cytometry constitutes an alternative for traditional methods of microorganisms identifi cation and analysis, including methods requiring cultivation step. It enables the detection of pathogens and other microorganisms contaminants without the need to culture microbial cells meaning that the sample (water, waste or food e.g. milk, wine, beer may be analysed directly. This leads to a signifi cant reduction of time required for analysis allowing monitoring of production processes and immediate reaction in case of contamination or any disruption occurs. Apart from the analysis of raw materials or products on different stages of manufacturing process, the fl ow cytometry seems to constitute an ideal tool for the assessment of microbial contamination on the surface of technological lines. Material and methods. In the present work samples comprising smears from 3 different surfaces of technological lines from fruit and vegetable processing company from Greater Poland were analysed directly with fl ow cytometer. The measured parameters were forward and side scatter of laser light signals allowing the estimation of microbial cell contents in each sample. Results. Flow cytometric analysis of the surface of food industry production lines enable the preliminary evaluation of microbial contamination within few minutes from the moment of sample arrival without the need of sample pretreatment. Conclusions. The presented method of fl ow cytometric initial evaluation of microbial state of food industry technological lines demonstrated its potential for developing a robust, routine method for the rapid and laborsaving detection of microbial contamination in food industry.

  12. Flow-Cytometric Isolation of Human Antibodies from a Nonimmune Saccharomyces cerevisiae Surface Display Library

    Feldhaus, Michael (BATTELLE (PACIFIC NW LAB)); Siegel, Robert W.(BATTELLE (PACIFIC NW LAB)); Opresko, Lee (BATTELLE (PACIFIC NW LAB)); Coleman, James R.(BATTELLE (PACIFIC NW LAB)); Feldhaus, Jane M.(BATTELLE (PACIFIC NW LAB)); Yeung, Yik A.(Massachusetts Institute Of Tec); Cochran, Jennifer R.(Massachusetts Institute Of Tec); Heinzelman, Peter (Massachusetts Institute Of Tec); Colby, David (Massachusetts Institute Of Tec); Swers, Jeffrey (Massachusetts Institute Of Tec); Graff, Christilyn (Massachusetts Institute Of Tec); Wiley, H Steven (BATTELLE (PACIFIC NW LAB)); Wittrup, K D.(Massachusetts Institute Of Tec)


    A nonimmune library of 109 human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow cytometric sorting. The yeast library can be amplified 1010-fold without measurable loss of clonal diversity, enabling effectively indefinite expansion of the library. The expression, stability, and antigen binding properties of more than 50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the utility of this approach for high throughput antibody isolation for proteomics applications.

  13. Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation.

    Weissenberg, R; Golan, R; Shochat, L; Lewin, L M


    The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.

  14. Multiplex competitive microbead-based flow cytometric immunoassay using quantum dot fluorescent labels

    Yu, Hye-Weon; Kim, In S. [School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju (Korea, Republic of); Niessner, Reinhard [Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universitaet Muenchen, Marchioninistrasse 17, 81377 Muenchen (Germany); Knopp, Dietmar, E-mail: [Chair for Analytical Chemistry, Institute of Hydrochemistry, Technische Universitaet Muenchen, Marchioninistrasse 17, 81377 Muenchen (Germany)


    Highlights: Black-Right-Pointing-Pointer First time, duplex competitive bead-based flow cytometric immunoassay was developed using ODs. Black-Right-Pointing-Pointer Antibody-coated QD detection probes and antigen-immobilized microspheres were synthesized. Black-Right-Pointing-Pointer The two model target analytes were low molecular weight compounds of microbial and chemical origin. Black-Right-Pointing-Pointer The determination of different water types was possible after simple filtration of samples. - Abstract: In answer to the ever-increasing need to perform the simultaneous analysis of environmental hazards, microcarrier-based multiplex technologies show great promise. Further integration with biofunctionalized quantum dots (QDs) creates new opportunities to extend the capabilities of multicolor flow cytometry with their unique fluorescence properties. Here, we have developed a competitive microbead-based flow cytometric immunoassay using QDs fluorescent labels for simultaneous detection of two analytes, bringing the benefits of sensitive, rapid and easy-of-manipulation analytical tool for environmental contaminants. As model target compounds, the cyanobacterial toxin microcystin-LR and the polycyclic aromatic hydrocarbon compound benzo[a]pyrene were selected. The assay was carried out in two steps: the competitive immunological reaction of multiple targets using their exclusive sensing elements of QD/antibody detection probes and antigen-coated microsphere, and the subsequent flow cytometric analysis. The fluorescence of the QD-encoded microsphere was thus found to be inversely proportional to target analyte concentration. Under optimized conditions, the proposed assay performed well within 30 min for the identification and quantitative analysis of the two environmental contaminants. For microcystin-LR and benzo[a]pyrene, dose-response curves with IC{sub 50} values of 5 {mu}g L{sup -1} and 1.1 {mu}g L{sup -1} and dynamic ranges of 0.52-30 {mu}g L{sup -1} and 0

  15. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Yazıcı, Serkan; Bülbül Başkan, Emel; Budak, Ferah; Oral, Barbaros; Adim, Şaduman Balaban; Ceylan Kalin, Zübeyde; Özkaya, Güven; Aydoğan, Kenan; Saricaoğlu, Hayriye; Tunali, Şükran


    We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression. PMID:26788525

  16. Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

    Serkan Yazıcı


    Full Text Available We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF. 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+, B cells (HLA-DR+, CD19+, and HLA-DR+CD19+, NKT cells (CD3+CD16+CD56+, and NK cells (CD3−CD16+CD56+. The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

  17. A Flow Cytometric and Computational Approaches to Carbapenems Affinity to the Different Types of Carbapenemases

    Pina-Vaz, Cidália; Silva, Ana P.; Faria-Ramos, Isabel; Teixeira-Santos, Rita; Moura, Daniel; Vieira, Tatiana F.; Sousa, Sérgio F.; Costa-de-Oliveira, Sofia; Cantón, Rafael; Rodrigues, Acácio G.


    The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computational analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes) were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem, or doripenem and killing kinetic curves performed with and without reinforcements of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3), a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incubation. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for carbapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable. PMID:27555844

  18. A flow cytometric and computational approaches to carbapenems affinity to the different types of carbapenemases

    Cidália Pina-Vaz


    Full Text Available The synergy of carbapenem combinations regarding Enterobacteriaceae producing different types of carbapenemases was study through different approaches: flow cytometry and computa-tional analysis. Ten well characterized Enterobacteriaceae (KPC, verona integron-encoded metallo-β-lactamases –VIM and OXA-48-like enzymes were selected for the study. The cells were incubated with a combination of ertapenem with imipenem, meropenem or doripenem and killing kinetic curves performed with and without reinforments of the drugs. A cephalosporin was also used in combination with ertapenem. A flow cytometric assay with DiBAC4-(3, a membrane potential dye, was developed in order to evaluate the cellular lesion after 2 h incuba-tion. A chemical computational study was performed to understand the affinity of the different drugs to the different types of enzymes. Flow cytometric analysis and time-kill assays showed a synergic effect against KPC and OXA-48 producing-bacteria with all combinations; only ertapenem with imipenem was synergic against VIM producing-bacteria. A bactericidal effect was observed in OXA-48-like enzymes. Ceftazidime plus ertapenem was synergic against ESBL-negative KPC producing-bacteria. Ertapenem had the highest affinity for those enzymes according to chemical computational study. The synergic effect between ertapenem and others carbapenems against different carbapenemase-producing bacteria, representing a therapeutic choice, was described for the first time. Easier and faster laboratorial methods for car-bapenemase characterization are urgently needed. The design of an ertapenem derivative with similar affinity to carbapenemases but exhibiting more stable bonds was demonstrated as highly desirable.

  19. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  20. Cytometric analysis of mammalian sperm for induced morphologic and DNA content errors

    Pinkel, D.


    Some flow-cytometric and image analysis procedures under development for quantitative analysis of sperm morphology are reviewed. The results of flow-cytometric DNA-content measurements on sperm from radiation exposed mice are also summarized, the results related to the available cytological information, and their potential dosimetric sensitivity discussed. (ACR)

  1. Standardizing flow cytometric assays in long-term population-based studies

    Melzer, Susanne; Bocsi, Jozsef; Tárnok, Attila


    Quantification of leukocyte subpopulations and characterization of antigen-expression pattern on the cellular surface can play an important role in diagnostics. The state of cellular immunology on the single-cell level was analyzed by polychromatic flow cytometry in a recent comparative study within the average Leipzig population (LIFE - Leipzig Research Centre for Civilization Diseases). Data of 1699 subjects were recorded over a long-time period of three years (in a total of 1126 days). To ensure compatibility of such huge data sets, quality-controls on many levels (stability of instrumentation, low intra-laboratory variance and reader independent data analysis) are essential. The LIFE study aims to analyze various cytometric pattern to reveal the relationship between the life-style, the environmental effects and the individual health. We therefore present here a multi-step quality control procedure for long-term comparative studies.

  2. Flow cytometric DNA analysis of ducks accumulating 137Cs on a reactor reservoir.

    George, L S; Dallas, C E; Brisbin, I L; Evans, D L


    The objective of this study was to detect red blood cell (rbc) DNA abnormalities in male, game-farm mallard ducks as they ranged freely and accumulated 137Cs (radiocesium) from an abandoned nuclear reactor cooling reservoir. Prior to release, the ducks were tamed to enable recapture at will. Flow cytometric measurements conducted at intervals during the first year of exposure yielded cell cycle percentages of DNA (G0/G1, S, G2 + M phases) of rbc, as well as coefficients of variation (CV) in the G0/G1 phase. DNA histograms of exposed ducks were compared with two sets of controls which were maintained 30 and 150 miles from the study site. 137Cs live wholebody burdens were also measured in these animals in a parallel kinetics study, and an approximate steady-state equilibrium was attained after about 8 months. DNA histograms from 2 of the 14 contaminated ducks revealed DNA aneuploid-like patterns after 9 months exposure. These two ducks were removed from the experiment at this time, and when sampled again 1 month later, one continued to exhibit DNA aneuploidy. None of the control DNA histograms demonstrated DNA aneuploid-like patterns. There were no significant differences in cell cycle percentages at any time point between control and exposed animals. A significant increase in CV was observed at 9 months exposure, but after removal of the two ducks with DNA aneuploidy, no significant difference was detected in the group monitored after 12 months exposure. An increased variation in the DNA and DNA aneuploidy could, therefore, be detected in duck rbc using flow cytometric analysis, with the onset of these effects being related to the attainment of maximal levels of 137Cs body burdens in the exposed animals.

  3. Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods

    M.O. de Jong (Marg); H. Rozemuller (Henk); J.G.J. Bauman (J. G J); J.W.M. Visser (Jan)


    textabstractThe main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that bind

  4. Biotinylation of interleukin-2 (IL-2) for flow cytometric analysis of IL-2 receptor expression. Comparison of different methods

    M.O. de Jong (Marg); H. Rozemuller (Henk); J.G.J. Bauman (J. G J); J.W.M. Visser (Jan)


    textabstractThe main prerequisites for the use of biotinylated ligands to study the expression of growth factor receptors on heterogeneous cell populations, such as peripheral blood or bone marrow, by flow cytometric methods, are that the biotinylated ligand retains its binding ability and that bind

  5. A short-term in vitro test for tumour sensitivity to adriamycin based on flow cytometric DNA analysis

    Engelholm, S A; Spang-Thomsen, M; Vindeløv, L L


    A new method to test the sensitivity of tumour cells to chemotherapy is presented. Tumour cells were incubated in vitro on agar, and drug-induced cell cycle perturbation was monitored by flow cytometric DNA analysis. In the present study the method was applied to monitor the effect of adriamycin...

  6. Clonal heterogeneity of small-cell anaplastic carcinoma of the lung demonstrated by flow-cytometric DNA analysis

    Vindeløv, L L; Hansen, H H; Christensen, I J


    Flow-cytometric DNA analysis yields information on ploidy and proliferative characteristics of a cell population. The analysis was implemented on small-cell anaplastic carcinoma of the lung using a rapid detergent technique for the preparation of fine-needle aspirates for DNA determination...

  7. Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

    Frita, R.; Rebelo, M.; Pamplona, A.; Vigario, A.M.; Mota, M.M.; Grobusch, M.P.; Haenscheid, T.


    Background: Malaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurat

  8. A simple and sensitive flow cytometric assay for determination of the cytotoxic activity of human natural killer cells

    Radosevic, Katarina; Radosevic, K.; Garritsen, Henk S.P.; Garritsen, H.S.P.; van Graft, M.; van Graft, Marja; de Grooth, B.G.; Greve, Jan


    A new, simple and sensitive flow cytometric assay for the determination of the cytotoxic activity of human natural killer cells is described. The assay is based on the use of two fluorochromes. The target cell population is stained with one fluorochrome (octadecylamine-fluorescein isothiocyanate,

  9. Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

    Fromm, Jonathan R; Kussick, Steven J; Wood, Brent L


    We demonstrate the feasibility of using flow cytometry (FC) to identify the Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Initial flow cytometric studies of the HRS cell line L1236 demonstrated potentially useful antigens for identifying HRS cells. L1236 cells spontaneously bound normal T cells, analogous to the T-cell rosetting of HRS cells seen in tissue sections of CHL, but these interactions could be blocked by using a cocktail of unlabeled antibodies to 4 adhesion molecules. Among 27 lymph nodes involved by CHL, FC enabled HRS cells to be identified in 89%, whereas none of 29 non-CHL neoplasms or 23 reactive lymph nodes demonstrated HRS populations. Of the CHL cases, 82% demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. Flow cytometric cell sorting experiments demonstrated typical HRS cytomorphologic features among the purified cells. FC may offer an alternative to immunohistochemical analysis in confirming the diagnosis of CHL in certain cases, and, through cell sorting, it provides a means of rapidly isolating pure HRS cells.

  10. Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage.

    Dertinger, Stephen D; Torous, Dorothea K; Hayashi, Makoto; MacGregor, James T


    The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring.

  11. Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

    Grobusch Martin P


    Full Text Available Abstract Background Malaria pigment (haemozoin, Hz has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs to assess antimalarial activity. Methods A standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine. Results A flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively

  12. A new spreadsheet method for the analysis of bivariate flow cytometric data

    Isacke Clare M


    Full Text Available Abstract Background A useful application of flow cytometry is the investigation of cell receptor-ligand interactions. However such analyses are often compromised due to problems interpreting changes in ligand binding where the receptor expression is not constant. Commonly, problems are encountered due to cell treatments resulting in altered receptor expression levels, or when cell lines expressing a transfected receptor with variable expression are being compared. To overcome this limitation we have developed a Microsoft Excel spreadsheet that aims to automatically and effectively simplify flow cytometric data and perform statistical tests in order to provide a clearer graphical representation of results. Results To demonstrate the use and advantages of this new spreadsheet method we have investigated the binding of the transmembrane adhesion receptor CD44 to its ligand hyaluronan. In the first example, phorbol ester treatment of cells results in both increased CD44 expression and increased hyaluronan binding. By applying the spreadsheet method we effectively demonstrate that this increased ligand binding results from receptor activation. In the second example we have compared AKR1 cells transfected either with wild type CD44 (WT CD44 or a mutant with a truncated cytoplasmic domain (CD44-T. These two populations do not have equivalent receptor expression levels but by using the spreadsheet method hyaluronan binding could be compared without the need to generate single cell clones or FACS sorting the cells for matching CD44 expression. By this method it was demonstrated that hyaluronan binding requires a threshold expression of CD44 and that this threshold is higher for CD44-T. However, at high CD44-T expression, binding was equivalent to WT CD44 indicating that the cytoplasmic domain has a role in presenting the receptor at the cell surface in a form required for efficient hyaluronan binding rather than modulating receptor activity. Conclusion

  13. The influence of fixation delay on mitotic activity and flow cytometric cell cycle variables.

    Bergers, E; Jannink, I; van Diest, P I; Cuesta, M A; Meyer, S; van Mourik, J C; Baak, J P


    Proliferation variables such as mitotic activity and the percentage of S-phase cells have been shown to be of prognostic value in many tumors, especially in breast cancer. However, some studies reported a decrease in mitotic activity caused by delay in fixation of the tissue. In contrast, other studies showed that the identifiability of mitotic figures decreases after fixation delay, but the total number of mitotic figures and also the percentage of S-phase cells remain unchanged. Most studies have been done on small numbers of experimental tumors, thus introducing the risk of selection bias. The aim of this study was to reinvestigate the influence of fixation delay on mitotic activity and cell cycle variables assessed by flow cytometry in an adequate number of resected human tissues to reach firmer conclusions. Resection specimens of 19 and 21 cases, respectively, for the mitotic activity estimate and the flow cytometric percentage of S-phase calculation were collected directly from the operating theater using lung, breast, and intestinal cancers and normal intestinal mucosa. The tissues were cut in pieces, and from each specimen, pieces were fixed in 4% buffered formaldehyde (for mitosis counting) as well as snap frozen (for flow cytometry) immediately after excision, as well as after a fixation delay of 1, 2, 4, 6, 8, 18, and 24 hours. Moreover, during the fixation delay, one series from each specimen was kept in the refrigerator and the second at room temperature. Thus, a total of 304 (19 X 16) and 336 (21 X 16) specimens were investigated for the mitotic activity estimate and the percentage of S-phase cells calculation, respectively. With regard to the estimation of the mitotic activity, both clear and doubtful mitotic figures were registered separately, obtaining an "uncorrected" and "corrected" (for doubtful mitotic figures) mitotic activity estimate. The percentage of S-phase cells was obtained by cell cycle analysis of flow cytometric DNA-histograms. The

  14. A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

    Gentry-Nielsen Martha J


    Full Text Available Abstract Background Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS to recruit PMNs to their lungs. They are then infected with live 5(-and 6 carboxyfluorescein diacetate succinimidyl ester (CFDA/SE labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria. Results The viability of the alveolar macrophages and PMNs isolated from the lavage fluid was >95%. The values of the percentage of PMNs in the lavage fluid as well as the percentage of PMNs associated with CFSE-labeled S. pneumoniae as measured through flow cytometry showed a high degree of correlation with the results from manual counting of cytospin slides. Conclusion This assay is suitable for measuring bacterial uptake within the infected lung. It can be adapted for use with other organisms and/or animal model systems.

  15. A novel flow cytometric hemozoin detection assay for real-time sensitivity testing of Plasmodium falciparum.

    Maria Rebelo

    Full Text Available Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz, which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50 were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.

  16. Evaluation of Red Cell Membrane Cytoskeletal Disorders Using a Flow Cytometric Method in South Iran

    Habib Alah Golafshan


    Full Text Available OBJECTIVE: The diagnosis of hereditary red blood cell (RBC membrane disorders, and in particular hereditary spherocytosis (HS and Southeast Asian ovalocytosis (SAO, is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5’-maleimide (EMA was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. METHODS: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI using a flow cytometer. RESULTS: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively, while macrocytic RBCs showed a significant increase in MFI (p<0.01. A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor. CONCLUSION: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

  17. Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.

    Gujar, Shashi A; Michalak, Tomasz I


    Woodchucks infected with woodchuck hepatitis virus (WHV) represent the closest natural animal model to study the immunopathogenesis of liver injury caused by essentially noncytopathic, highly human specific hepatitis B virus (HBV). The importance of antiviral T cell response in induction of hepatitis and in control of HBV replication has been demonstrated. However, the understanding of how these responses contribute to the development of different immunomorphological forms of liver disease and their outcomes remain elusive. In this study, we established and standardized a flow cytometry assay using peripheral blood mononuclear cells labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) to assess WHV-specific and mitogen-driven T lymphocyte proliferative responses in woodchucks. The assay is of significantly greater sensitivity than the adenine incorporation assay currently used when applied to measure either WHV-specific T cell responses in acute (P measuring cell division rates. The study shows that woodchuck PBMC labeled with CFSE exhibit light scatter and fluorescence profiles compatible to those of human PBMC, allowing quantitation and deconvolution of the flow cytometric data by applying the existing analytical softwares. The availability of this novel assay should facilitate a more precise and comprehensive evaluation of hepadnavirus-specific and generalized T cell responses in experimental WHV hepatitis.

  18. Flow cytometric analysis of oil palm: a preliminary analysis for cultivars and genomic DNA alteration

    Warawut Chuthammathat


    Full Text Available DNA contents of oil palm (Elaeis guineensis Jacq. cultivars were analyzed by flow cytometry using different external reference plant species. Analysis using corn (Zea mays line CE-777 as a reference plant gave the highest DNA content of oil palm (4.72±0.23 pg 2C-1 whereas the DNA content was found to be lower when using soybean (Glycine max cv. Polanka (3.77±0.09 pg 2C-1 or tomato (Lycopersicon esculentum cv. Stupicke (4.25±0.09 pg 2C-1 as a reference. The nuclear DNA contents of Dura (D109, Pisifera (P168 and Tenera (T38 cultivars were 3.46±0.04, 3.24±0.03 and 3.76±0.04 pg 2C-1 nuclei, respectively, using soybean as a reference. One haploid genome of oil palm therefore ranged from 1.56 to 1.81±109 base pairs. DNA contents from one-year-old calli and cell suspension of oil palm were found to be significantly different from those of seedlings. It thus should be noted that genomic DNA alteration occurred in these cultured tissues. We therefore confirm that flow cytometric analysis could verify cultivars, DNA content and genomic DNA alteration of oil palm using soybean as an external reference standard.

  19. Comparison of multidimensional flow cytometric data by a novel data mining technique

    Leary, James F.; Smith, Jacob; Szaniszlo, Peter; Reece, Lisa M.


    Most flow/image cytometric data analysis methods look for clusters in the data corresponding to specific cell subpopulations. Comparisons between different cytometry datafiles often use human pattern recognition visualization of all the different combinations of variables ("parameters") two at a time in so-called bivariate scattergrams. Not only is this tedious, but it can miss potential clusters due to projection of higher dimensional dataspaces down onto two dimensional planes making them indiscernible as separate clusters. Novel data mining algorithms, implemented in software allow for the comparison of two or more higher dimensional datafiles without the requirement for reduction of dimensionality for human visualization. Of equal importance is the comparison of higher dimensional clusters which may move around slightly in space, yet still be "similar" according to algorithms which provide measures of similarity. This software, written in C/C++ and currently implemented in software with a Windows graphical user interface, allows for direct reading of FCS2.0 format flow cytometry datafiles of any number of parameters. In a few minutes or less, complex multiparameter data of two or more files can be compared on a personal computer or workstation. The software operates in either supervised or unsupervised mode, depending on whether the user wishes to include prior user knowledge or in a data mining discovery mode. Differences between these files can be exported as sub-datafiles which can be further analyzed using any other software that can read FCS2.0 data format.

  20. A new flow cytometric method for quantitative assessment of lymphocyte mitogenic potentials.

    Yamamura, Y; Rodriguez, N; Schwartz, A; Eylar, E; Bagwell, B; Yano, N


    A new flow cytometric method was developed to quantitatively assess lymphocyte proliferation simultaneously for different subsets. The cells were stained with a fluorescent dye, PKH-26 and were stimulated with mitogens. The fluorescence intensities (FL2) of proliferating cells were measured by flow cytometry; and each subset was identified by the use of a monoclonal antibody (Mab)-fluorescein-isothiocyanate (FITC) (FL1). FL2 histograms were then analyzed by the cell proliferation model based on the ModFit software (Verity). This new method revealed information which could not be obtained by conventional mitogen assays. For example, the CD4+ and the CD4- T-subsets responded to phytohemagglutinin (PHA) quite differently from each other and it was indicated that activation of one population could significantly alter the response of the other. In addition, even within a subset, all activated cells did not proliferate uniformly. Some cells divided only once while others underwent further cellular division during the same time period. The method is, therefore, invaluable for studying the nature and the extent of interactions between different cellular subsets within a culture.

  1. Flow cytometric evaluation of the intracellular bacterium, Wolbachia pipientis, in mosquito cells

    Fallon, Ann M


    Wolbachia is an obligate intracellular bacterium (Anaplasmataceae, Rickettisales) that occurs in arthropods and filarial worms, and spreads by vertical transmission in the oocyte cytoplasm. In insects, reproductive distortions associated with Wolbachia, such as cytoplasmic incompatibility in mosquitoes, have potential value for controlling pests, including species that transmit human, animal and plant diseases. Wolbachia strains that propagate as a persistent infection in insect cell lines provide an important resource for developing the genetic tools that will facilitate these applications. Here I describe conditions for flow cytometric evaluation of Wolbachia growth in persistently infected mosquito cells. Cytometry parameters were established using uninfected mosquito cells and Escherichia coli as a surrogate for Wolbachia, and quantitation was correlated with cell counts determined with a Coulter electronic cell counter and bacterial counts based on optical density. The protocol was validated by showing depletion of Wolbachia in medium containing tetracycline and rifampicin, and sensitivity of Wolbachia to treatment of host cells with paraquat, an oxidizing agent, and lumiflavin, an inhibitor of riboflavin uptake. The Wolbachia peak on the flow cytometry histogram was shown to contain Wolbachia by DNA analysis using the polymerase chain reaction, and by infection of naive recipient cells. This approach will streamline investigation of Wolbachia growth in insect cell lines and facilitate identification of culture conditions that select for Wolbachia-infected cells. PMID:25300665

  2. Flow cytometric analysis using SYBR Green I for genome size estimation in coffee.

    Ronildo Clarindo, Wellington; Roberto Carvalho, Carlos


    Plant genome size has been measured by flow cytometry using propidium iodide as a dye for nuclear DNA staining. However, some authors have reported the occurrence of genome size estimation errors, especially in plants rich in secondary metabolites, such as the coffee tree. In this context, we tested an alternative cytometric protocol using the SYBR Green I as a fluorochrome for stoichiometrically staining nuclear double-stranded DNA in Coffea canephora (2x) and Coffea arabica (4x). The results showed that the respective mean genome size measured from nuclei stained with SYBR Green I and propidium iodide was statistically identical. However, the G(0)/G(1) peaks of nuclei stained with SYBR Green I exhibited lower coefficient variations (1.57-2.85%) compared to those stained with propidium iodide (2.75-4.80%). Coefficient variation statistical data suggest that SYBR Green I is adequate for stoichiometric nuclei staining using this methodology. Our results provide evidence that SYBR Green I can be used in flow cytometry measurements of plants, with the advantages of minimizing errors in nuclear DNA content quantification, staining relatively quicker, with high affinity, and being less mutagenic than propidium iodide.

  3. Flow cytometric sorting coupled with exon capture sequencing identifies somatic mutations in archival lymphoma tissues.

    Jiang, Nenggang; Chen, Christopher; Gong, Qiang; Shields, Kristen; Li, Yuping; Chen, YuanYuan; Song, Joo; McKeithan, Timothy W; Chan, Wing C


    The enormous number of archived formalin-fixed paraffin-embedded (FFPE) tissues available are a valuable resource of material for research. However, the use of such tissues poses many challenges, among which is the difficulty of isolating different cell populations within the tissue. In this study, we used tissue from two types of non-Hodgkin lymphoma as a model to demonstrate a method we have established and optimized to separate FFPE samples into distinct tumor and nonmalignant populations. Using FFPE reactive tonsil sections, various approaches for antigen retrieval and labeling, and the effectiveness of flow cytometric sorting were tested. We found that, among the 11 cell surface or intracellular antigen markers investigated, CD3ɛ, CD79A, LAT, PD-1, and PAX5 could be successfully labeled after antigen retrieval in Tris-EDTA buffer (pH 8.0) at 65 °C for 60 min, and 1.8-2.7 μg DNA per million cells could be extracted after sorting with DNA quality similar to that of tissue without staining or sorting. To test whether we could perform next-generation sequencing using a custom capture platform on sorted cells, we used three lymphoma cases with FFPE tissues which had been stored for 1 to 4 years. We demonstrated that the DNA from sorted cells was adequate for exon capture sequencing. By comparing the sequencing results between neoplastic and normal populations, somatic mutations could be clearly identified in the tumor population with variant frequencies as low as 11.7%.The corresponding normal fraction clearly helps in the analysis of somatic mutations and the exclusion of artifacts. This study provides an approach using flow cytometric sorting to separate different cellular populations in paraffin-embedded tissues and to unambiguously distinguish somatic mutations from germline variants or artifacts. This approach is also useful in enriching the tumor component in samples with heterogeneous components and low tumor content.Laboratory Investigation advance

  4. FlowFP: A Bioconductor Package for Fingerprinting Flow Cytometric Data

    Wade T. Rogers; Herbert A. Holyst


    A new software package called flowFP for the analysis of flow cytometry data is introduced. The package, which is tightly integrated with other Bioconductor software for analysis of flow cytometry, provides tools to transform raw flow cytometry data into a form suitable for direct input into conventional statistical analysis and empirical modeling software tools. The approach of flowFP is to generate a description of the multivariate probability distribution function of flow cytometry data i...

  5. Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

    Talarmin, A.; van Wambeke, F.; Catala, P.; Courties, C.; Lebaron, P.


    The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l-1 h-1 (n=30). Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok) were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA) with high scatter (HNA-hs) and low scatter (HNA-ls) and low nucleic acid containing cells (LNA). Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell-1 h-1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30). In surface layers (from 0 m down to the deep chlorophyll depth, DCM), cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively). Nevertheless, on average HNA-hs (26%) and LNA (27%) equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell-1 h-1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The mixotrophic features of certain photosynthetic prokaryotes and the high contribution of LNA cells to leucine incorporation within the microbial

  6. Flow cytometric assessment of specific leucine incorporation in the open Mediterranean

    A. Talarmin


    Full Text Available The surface of the Mediterranean Sea is a low-phosphate-low-chlorophyll marine area where marine heterotrophic prokaryotes significantly contribute to the biogeochemical cycles of all biogenic elements such as carbon, notably through the mineralization of dissolved organic compounds. Cell-specific leucine incorporation rates were determined in early summer in the open stratified Mediterranean Sea. The bulk leucine incorporation rate was on average 5 ± 4 pmol leu l−1 h−1 (n=30. Cell-specific 3H-leucine incorporation rates were assayed using flow cytometry coupled to cell sorting. Heterotrophic prokaryotes (Hprok were divided into cytometric groups according to their side scatter and green fluorescence properties: high nucleic acid containing cells (HNA with high scatter (HNA-hs and low scatter (HNA-ls and low nucleic acid containing cells (LNA. Cell-specific leucine incorporation rates of these cytometric groups ranged from 2 to 54, 0.9 to 11, and 1 to 12 × 10-21 mol cell−1 h−1, respectively. LNA cells represented 45 to 63% of the Hprok abundance, and significantly contributed to the bulk leucine incorporation rates, from 12 to 43%. HNA/LNA ratios of cell-specific leucine incorporation were on average 2.0 ± 0.7 (n=30. In surface layers (from 0 m down to the deep chlorophyll depth, DCM, cell-specific rates of HNA-hs were elevated (7 and 13 times greater than LNA and HNA-ls, respectively. Nevertheless, on average HNA-hs (26% and LNA (27% equally contributed to the bulk leucine incorporation in these layers. Prochlorococcus cells were easily sorted near the DCM and displayed cell-specific leucine incorporation rates ranging from 3 to 55 × 10-21 mol leu cell−1 h−1, i.e. as high as HNA-hs'. These sorted groups could therefore be defined as key-players in the process of leucine incorporation into proteins. The

  7. [Flow cytometric analysis of ICRF-193 influence on cell passage through mitosis].

    Shatrova, A N; Aksenov, N D; Zenin, V V


    Studying the effect of topoisomerase II (topo II) inhibitors on cell passage through mitosis seems to be important for understanding the role of this enzyme during chromosome condensation and segregation. A flow cytometric assay (Zenin et al., 2001) allowed to determine the mitotic index, and to discriminate between not only cells in G2 and M phases (including metaphase and anaphase cells), but also cells in pseudo-G1 with 4c DNA content. It is shown that topo II catalytic inhibitor ICRF-193 blocks G2-M transition in a lymphoblastoid cell line GM-130. Addition of caffeine to cells abrogated a block of their entering mitosis but not the inhibitor action. Cells entered mitosis, which was proven by the presence of chromosomes in the examined specimen, and, bypassing anaphase, appeared in pseudo-G1 with 4c DNA content. We have found that in the presence of ICRF-193 cells, GM-130 and Hep-2 lines, previously blocked by nocodazole when in mitosis and then washed, pass through metaphase, enter anaphase and leave it to pass to pseudo-G1 with the 4c DNA content. Thus, by inhibiting topo II activity ICRF-193 causes abnormal mitotic transition.

  8. Establishment of multiplexed, microsphere-based flow cytometric assay for multiple human tumor markers

    Kai SUN; Qian WANG; Xiao-hui HUANG; Mao-chuan ZHEN; Wen LI; Long-juan ZHANG


    Aim: The multiplexed, microsphere-based flow cytometric assay (MFCA) for mul- tiple human tumor markers was established for the early screening and detection of suspected cancer patients. Methods: Covalent coupling of capture antibodies directed against their respective tumor markers to fluorescent microspheres was performed by following the protocols recommended by a commercial corporation with some modifications. The coupling efficiency and cross-reactivity were iden- tified by the Luminex 100 system and associated software. The standard curve was constructed by using serial dilution of recombinant tumor marker standards and was validated by comparison with ELISA for quantifying the tumor markers in serum samples. Results: The identifications revealed that the coupling proce- dures were successful without non-specific cross-reactivity and the standard curve was highly efficient. However, it was necessary to ensure the quality con- trol of the coupling process since slight variations in the coupling procedures could profoundly affect the density of capture reagents coupled to the microspheres and consequently adversely affect the assay precision. In addition to its multi-analyte capability, the MFCA system had definite advantages, such as higher reproducibility, greater dynamic range of measurement, and considerably less preparation time and labor over the conventional "gold standard", which was the ELISA. Conclusion: The successful establishment of the MFCA system for the simultaneous detection of multiple tumor markers will provide the foundation for the further study of clinical applications.

  9. Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans

    Jesús M. Egido


    Full Text Available We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripherial blood (HCPB, using commercially available phycoerythrin-conjugated latex particles of 1µm diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984 standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripherial blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB.Se da cuenta de un método rápido para la cuantización del flujo citométrico de la fagocitosis en sangre periférica completamente heparinizada (HCPB, mediante la utilización de partículas de látex phycoerythrin-conjugadas de 1µm de diámetro disponibles comercialmente. El método es más rápido y presenta mayor reproducibilidad que la técnica estandar de Bjerknes' (1984 utilizando propidium iodide-teñida Candida albicans, aplicada convencionalmente a la capa leucocitica de sangre periférica pero modificada por HCPB. Tambien damos cuenta de una modificación de Bjerknes' Intracellular Killing Test para permitir su aplicación a HCPB.

  10. Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

    Fujimura, Masato; Masuda, Kenichi; Hayashiya, Makio; Okayama, Taro


    Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (Pfood allergens may be involved in the pathogenesis of canine FA.

  11. A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin

    Maass, Katie F.; Kulkarni, Chethana; Quadir, Mohiuddin A.; Hammond, Paula T.; Betts, Alison M.; Wittrup, K. Dane


    Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell’s ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 105 – 1010 doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell’s ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4 – 12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug’s single-cell potency and can be used for any fluorescent or fluorescently-labeled drug, including nanoparticles or antibody-drug conjugates. PMID:26344409

  12. Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

    Coignoul Freddy


    Full Text Available Abstract Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction.

  13. Antiphospholipid antibody syndrome: the flow cytometric annexin A5 competition assay as a diagnostic tool.

    Tomer, A; Bar-Lev, S; Fleisher, S; Shenkman, B; Friger, M; Abu-Shakra, M


    The mechanism underlying hypercoagulability in antiphospholipid antibody syndrome (APS) is uncertain. Here, we present a flow-cytometric assay (FCA) based on the hypothesis that anti-platelet-anionic-phospholipid autoantibodies (aPL) interfere with the activity of the natural anticoagulant protein annexin A5, thereby accelerating platelet procoagulant activity. This study assessed the clinical utility of the feasible FCA, which demonstrates the competition of the patient's aPL with the binding of annexin A5 to the platelet-anionic-phospholipids, in the diagnosis of APS. Sixty-two (94%) of 66 APS patients, 20 (51%) of 39 patients with systemic lupus erythematosus and two (4%) of 49 healthy individuals were positive by FCA. Compared with the anticardiolipin (aCL) assay, the relative sensitivity was 82% and the specificity 73.3%. However, 19 (25%) aCL-negative patients were positive by FCA; 12 were positive for lupus-anticoagulant (LA). Compared with LA assay, the relative sensitivity was 85% and the specificity 72.2%. However, 21 (26%) LA-negative patients were FCA-positive, 12 were positive for aCL. The FCA was particularly sensitive for APS patients with arterial (97.0%) and gestational vascular complications (100%) with overall sensitivity of 95% and specificity of 97%. Our findings suggest that the FCA is practical, sensitive and specific for the detection of clinically relevant aPL in the diagnosis of APS.

  14. Dissociation of skeletal muscle for flow cytometric characterization of immune cells in macaques.

    Liang, Frank; Ploquin, Aurélie; Hernández, José DelaO; Fausther-Bovendo, Hugues; Lindgren, Gustaf; Stanley, Daphne; Martinez, Aiala Salvador; Brenchley, Jason M; Koup, Richard A; Loré, Karin; Sullivan, Nancy J


    The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classic human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provide a basis for defining immediate post-injection vaccine responses in primates.

  15. Successful low dose insemination of flow cytometrically sorted ram spermatozoa in sheep.

    de Graaf, S P; Evans, G; Maxwell, W M C; Downing, J A; O'Brien, J K


    The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo SX) and cryopreservation was assessed after low-dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen-impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 x 10(6), 5 x 10(6) or 15 x 10(6) motile sorted frozen-thawed (S(1), S(5), or S(15) respectively) or non-sorted frozen-thawed (C(1), C(5) or C(15) respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 x 10(6) motile non-sorted frozen-thawed spermatozoa (C(50)) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C(50) (24/38, 63.2%), C(15) (37/54, 68.5%), S(15) (38/57, 66.7%), S(5) (37/56, 66.1%) and S(1) (32/52, 61.5%) groups (p > 0.05), but lower for C(5) (19/48, 39.6%) and C(1) (19/55, 34.5%) treatments (p sheep as a reduction in the minimum effective sperm number will allow a corresponding decrease in the associated cost per dose.

  16. Flow cytometric viability assessment of lactic acid bacteria starter cultures produced by fluidized bed drying.

    Bensch, Gerald; Rüger, Marc; Wassermann, Magdalena; Weinholz, Susann; Reichl, Udo; Cordes, Christiana


    For starter culture production, fluidized bed drying is an efficient and cost-effective alternative to the most frequently used freeze drying method. However, fluidized bed drying also poses damaging or lethal stress to bacteria. Therefore, investigation of impact of process variables and conditions on viability of starter cultures produced by fluidized bed drying is of major interest. Viability of bacteria is most frequently assessed by plate counting. While reproductive growth of cells can be characterized by the number of colony-forming units, it cannot provide the number of viable-but-nonculturable cells. However, in starter cultures, these cells still contribute to the fermentation during food production. In this study, flow cytometry was applied to assess viability of Lactobacillus plantarum starter cultures by membrane integrity analysis using SYBR®Green I and propidium iodide staining. The enumeration method established allowed for rapid, precise and sensitive determination of viable cell concentration, and was used to investigate effects of fluidized bed drying and storage on viability of L. plantarum. Drying caused substantial membrane damage on cells, most likely due to dehydration and oxidative stress. Nevertheless, high bacterial survival rates were obtained, and granulates contained in the average 2.7 × 10(9) viable cells/g. Furthermore, increased temperatures reduced viability of bacteria during storage. Differences in results of flow cytometry and plate counting suggested an occurrence of viable-but-nonculturable cells during storage. Overall, flow cytometric viability assessment is highly feasible for rapid routine in-process control in production of L. plantarum starter cultures, produced by fluidized bed drying.

  17. An imaging flow cytometric method for measuring cell division history and molecular symmetry during mitosis.

    Filby, Andrew; Perucha, Esperanza; Summers, Huw; Rees, Paul; Chana, Prabhjoat; Heck, Susanne; Lord, Graham M; Davies, Derek


    Asymmetric cell division is an important mechanism for generating cellular diversity, however, techniques for measuring the distribution of fate-regulating molecules during mitosis have been hampered by a lack of objectivity, quantitation, and statistical robustness. Here we describe a novel imaging flow cytometric approach that is able to report a cells proliferative history and cell cycle position using dye dilution, pH3, and PI staining to then measure the spatial distribution of fluorescent signals during mitosis using CCD-derived imagery. Using Jurkat cells, resolution of the fluorescently labeled populations was comparable to traditional PMT based cytometers thus eliminating the need to sort cells with specific division histories for microscopy. Subdividing mitotic stages by morphology allowed us to determine the time spent in each cell cycle phase using mathematical modeling approaches. Furthermore high sample throughput allowed us to collect statistically relevant numbers of cells without the need to use blocking agents that artificially enrich for mitotic events. The fluorescent imagery was used to measure PKCζ protein and EEA-1+ endosome distribution during different mitotic phases in Jurkat cells. While telophase cells represented the favorable population for measuring asymmetry, asynchronously dividing cells spent approximately 43 seconds in this stage, explaining why they were present at such low frequencies. This necessitated the acquisition of large cell numbers. Interestingly we found that PKCζ was inherited asymmetrically in 2.5% of all telophasic events whereas endosome inheritance was significantly more symmetrical. Furthermore, molecular polarity at early mitotic phases was a poor indicator of asymmetry during telophase highlighting that, though rare, telophasic events represented the best candidates for asymmetry studies. In summary, this technique combines the spatial information afforded by fluorescence microscopy with the statistical

  18. Flow cytometric analysis of lectin binding to in vitro-cultured Perkinsus marinus surface carbohydrates

    Gauthier, J.D.; Jenkins, J.A.; La Peyre, Jerome F.


    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. ?? American Society of Parasitologists 2004.

  19. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).

    Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S


    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  20. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki


    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  1. Evaluation of the S phase distribution of flow cytometric DNA histograms by autoradiography and computer algorithms.

    Sheck, L E; Muirhead, K A; Horan, P K


    Cell sorting and tritiated thymidine autoradiography were used to define the distribution of S phase cells in flow cytometric DNA histograms obtained from exponential mouse lymphoma cells (L5178Y). The numbers of labeled S phase cells, autoradiographically determined from cells sorted at 2-channel intervals in the G1/early S and late S/G2M regions of the histogram, were compared with the numbers of computed S phase cells in comparable 2-channel intervals as predicted by several computer algorithms used to extract cell cycle phase distributions from DNA histograms. Polynomial and multirectangle algorithms gave computed estimates of total %S in close agreement with the tritiated thymidine labeling index for the cell population, while multi-Gaussian algorithms underestimated %S. Interval autoradiographic and algorithm studies confirmed these results in that no significant differences were found between the autoradiographic S phase distribution and S phase distributions calculated by the polynomial and multirectangle models. However, S phase cells were significantly underestimated in G1/early S by a constrained multi-Gaussian model and in both G1/early S and late S/G2 by an unconstrained multi-Gaussian model. For the particular cell line (L5178Y), staining protocol (mithramycin following ethanol fixation) and instrumentation (Coulter TPS-2 cell sorter) used in this study, close agreement between computed %S and tritiated thymidine labeling index was found to be a reliable indicator of an algorithm's success in resolving S phase cells in the G1/S and S/G2 transition regions of the DNA histograms.

  2. Flow Cytometric Assessment of Bacterial Abundance in Soils, Sediments and Sludge.

    Frossard, Aline; Hammes, Frederik; Gessner, Mark O


    Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz(®) solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high.

  3. Novel nuclei isolation buffer for flow cytometric genome size estimation of Zingiberaceae: a comparison with common isolation buffers.

    Sadhu, Abhishek; Bhadra, Sreetama; Bandyopadhyay, Maumita


    Cytological parameters such as chromosome numbers and genome sizes of plants are used routinely for studying evolutionary aspects of polyploid plants. Members of Zingiberaceae show a wide range of inter- and intrageneric variation in their reproductive habits and ploidy levels. Conventional cytological study in this group of plants is severely hampered by the presence of diverse secondary metabolites, which also affect their genome size estimation using flow cytometry. None of the several nuclei isolation buffers used in flow cytometry could be used very successfully for members of Zingiberaceae to isolate good quality nuclei from both shoot and root tissues. The competency of eight nuclei isolation buffers was compared with a newly formulated buffer, MB01, in six different genera of Zingiberaceae based on the fluorescence intensity of propidium iodide-stained nuclei using flow cytometric parameters, namely coefficient of variation of the G0/G1 peak, debris factor and nuclei yield factor. Isolated nuclei were studied using fluorescence microscopy and bio-scanning electron microscopy to analyse stain-nuclei interaction and nuclei topology, respectively. Genome contents of 21 species belonging to these six genera were determined using MB01. Flow cytometric parameters showed significant differences among the analysed buffers. MB01 exhibited the best combination of analysed parameters; photomicrographs obtained from fluorescence and electron microscopy supported the superiority of MB01 buffer over other buffers. Among the 21 species studied, nuclear DNA contents of 14 species are reported for the first time. Results of the present study substantiate the enhanced efficacy of MB01, compared to other buffers tested, in the generation of acceptable cytograms from all species of Zingiberaceae studied. Our study facilitates new ways of sample preparation for further flow cytometric analysis of genome size of other members belonging to this highly complex polyploid family.

  4. Effect of intra-cellular trafficking on flow cytometric measurement of neutrophil's oxidative status in iron deficient pregnant females.

    Youssef, Soha R; Hendawy, Sherif F; Boshnak, Noha H; Sedhom, Mariana S


    Iron deficiency and iron deficiency anemia are prevalent among pregnant women particularly in developing countries. This study aimed to evaluate the iron status among Egyptian pregnant women and its impact on their neutrophil's count and antimicrobial functions. Ninety pregnant females underwent complete blood count, iron profile, flow cytometric studies for neutrophil myeloperoxidase expression & oxidative burst using dihydrorhodamine 123 (DHR) after phorbol-12-myristate-13-acetate (PMA) stimulation as well as neutrophil phagocytic and lytic indices. According to percent saturation 54/90 women (60%) were iron deficient (women were in their third trimester compared to controls. No significant difference was found between the iron deficient & sufficient groups as regards anemia despite a positive correlation between haemoglobin level and percent saturation (P=.02). Both the phagocytic and lytic indices were significantly lower among the cases compared to controls (P=.014 & .002 respectively). Cases and controls were comparable as regards flow cytometric studies of neutrophils' myeloperoxidase and oxidative burst (P>.05). No significant correlation was found between any of the iron profile parameters and the oxidative burst by flow cytometry. Functional microphage assay (phagocytic and lytic indices) may be more relevant and cost effective than flow cytometry assays of myeloperoxidase and oxidative burst in reflecting either iron status or cellular immunity in pregnancy. © 2017 Wiley Periodicals, Inc.

  5. Effects of Hoechst33342 staining on the viability and flow cytometric sex-sorting of frozen-thawed ram sperm.

    Quan, Guo Bo; Ma, Yuan; Li, Jian; Wu, Guo Quan; Li, Dong Jiang; Ni, Yi Na; Lv, Chun Rong; Zhu, Lan; Hong, Qiong Hua


    Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 μM, 120 μM, 160 μM, 200 μM, 240 μM, or 320 μM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 μM Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (Pram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two sperm populations with X and Y chromosome when the Hoechst33342 concentration was 160 μM. Moreover, when the staining duration was equal to or longer than 45 min, the frozen-thawed sperm can be successfully sorted in the presence of 160μM Hoechst33342. In conclusion, Hoechst33342 staining can detrimentally influence viability of frozen-thawed ram sperm except acrosome and in vitro

  6. Postlarval muscle growth in fish: a DNA flow cytometric and morphometric analysis.

    Alfei, L; Maggi, F; Parvopassu, F; Bertoncello, G; De Vita, R


    The mechanism of postlarval fish myotomal growth was investigated in trout (Salmo gairdneri) by means of morphometric and cytofluorometric analysis. The mechanism by which new fibres are added during postlarval growth (hyperplasia) is not fully understood. In histological cross sections these new fibres have a small diameter which give the muscle a "mosaic" appearance. One hypothesis suggested that they could be derived from the proliferative activity of satellite cells. DNA cytofluorometric analysis of nuclei suspensions obtained from trout white myotomal muscle during different developmental stages (eleutherembyronic; alevin; yearling and adult) showed a consistently low S-cytometric phase during all stage in which myofibres of small diameters were present. The percentage of such small fibres, determined by morphometric analysis, suggested that satellite cells are the proliferative population. In fact, their percentages, as determined by morphometric analysis in histological section, bear a linear relationship with the S-cytometric phase percent nuclei (R = 0.927). Only in adults (67 cm in size) there was a significant decrease in the S-cytometric phase. At this stage, in histological sections, the myotomal muscle no longer had a "mosaic" appearance because of the disappearance of the small fibres. It may, therefore, be supposed that in the cm 67 adult specimens, the proliferative population is entering the G0 phase. It is known, in fact, that muscle growth proceeds only by fibre hypertrophy in trout longer than 70 cm in length (Stickland, 1983).

  7. Correlation between histological grading and ploidy status in potentially malignant disorders of the oral mucosa: A flow cytometric analysis

    T Vijayavel


    Full Text Available Background: Histopathological grading of oral dysplastic lesions is the method of choice for evaluating malignant and potentially malignant disorders. Owing to inter- and intra-observer variability, determination of the DNA ploidy status of lesions may serve as an adjunct in the prediction of malignant transformation. Aim: To correlate histopathological grading and ploidy status in potentially malignant and malignant disorders of the oral mucosa. Settings and Design: A pilot study was done with 30 patients (10 patients with oral potentially malignant disorders predominantly leukoplakia, 10 patients with oral malignant lesions and 10 patients with normal mucosa. Materials and Methods: Incisional biopsy was done after isolating the biopsy site with 1% Toluidine blue staining. Two sections of the tissue were removed and sent for histopathological and Flow-cytometric analysis respectively. Histopathological diagnosis was obtained and compared with Flow-cytometric results which were graded as diploid and aneuploid. Further, the S - phase fraction, DNA index were also calculated to evaluate the severity of malignant transformation or malignancy. Statistical Analysis: The results were analyzed using Pearson Chi-Square Test. Results: There exists a significant correlation between histopathology and ploidy status in both potentially malignant and malignant group. (P = 0.002. Conclusion: The data from this study has shown that DNA Ploidy analysis can be used as a valuable tool in assessing the carcinomatous progression of potentially malignant and malignant lesions.

  8. A sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages

    Mosser David M


    Full Text Available Abstract Background The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay. Methods To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 × 106 cells/mL. Leishmania (Leishmania chagasi parasites (stationary-phase were adjusted to 5 × 107 cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18 antibodies and analyzed by flow citometry. Results Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1 β2 integrin. Conclusion Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.

  9. A cluster analysis method for identification of subpopulations of cells in flow cytometric list-mode arrays

    Li, Z. K.


    A specialized program was developed for flow cytometric list-mode data using an heirarchical tree method for identifying and enumerating individual subpopulations, the method of principal components for a two-dimensional display of 6-parameter data array, and a standard sorting algorithm for characterizing subpopulations. The program was tested against a published data set subjected to cluster analysis and experimental data sets from controlled flow cytometry experiments using a Coulter Electronics EPICS V Cell Sorter. A version of the program in compiled BASIC is usable on a 16-bit microcomputer with the MS-DOS operating system. It is specialized for 6 parameters and up to 20,000 cells. Its two-dimensional display of Euclidean distances reveals clusters clearly, as does its 1-dimensional display. The identified subpopulations can, in suitable experiments, be related to functional subpopulations of cells.

  10. Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41.

    do Céu Monteiro, M; Sansonetty, F; Gonçalves, M J; O'Connor, J E


    Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca2+ is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca2+ mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Anticoagulated blood was diluted in Tyrode's buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41+ events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and time-dependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. In these conditions, a very fast and transient increase of cytosolic-free Ca2+ was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca2+ levels. Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy.

  11. A Protocol for the Comprehensive Flow Cytometric Analysis of Immune Cells in Normal and Inflamed Murine Non-Lymphoid Tissues.

    Yu, Yen-Rei A; O'Koren, Emily G; Hotten, Danielle F; Kan, Matthew J; Kopin, David; Nelson, Erik R; Que, Loretta; Gunn, Michael D


    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions.

  12. Flow cytometric analyses of the viability, surface marker expression and function of lymphocytes from children following cryopreservation.

    Chen, Xi; Zhang, Hui; Mou, Wenjun; Qi, Zhan; Ren, Xiaoya; Wang, Guoliang; Jiao, Hong; Kong, Xiaohui; Gui, Jingang


    Flow cytometric analysis is important for the investigation and clinical preparation of lymphocytes from children. However, the strict requirement of cell freshness and inter‑assay variability limits the application of this methodology for pediatric investigations. Therefore, it is necessary to identify a reliable cryopreservative method capable of maintaining high cell viability and proper cell function in lymphocytes from children. In the present study, eight commonly‑used cell cyropreservative methods were used, and their effects on cell viability, surface marker expression and cell function were examined. In addition, how these methods affect the distribution of T‑cell receptor Vβ subfamilies were also determined. The results of the present study provided valuable experimental evidence, based on which the optimal method for the cryopreservation of lymphocytes from children in pediatric investigations and clinical applications can be selected.

  13. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    López-Carro Beatriz


    Full Text Available Abstract Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson. This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.

  14. Ultra-Fast and Optimized Method for the Preparation of Rodent Testicular Cells for Flow Cytometric Analysis

    Rodríguez-Casuriaga Rosana


    Full Text Available Abstract Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson. This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use.

  15. Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis.

    Odhiambo, J F; Sutovsky, M; DeJarnette, J M; Marshall, C; Sutovsky, P


    The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L'Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P flow cytometric semen evaluation.

  16. Establishing the flow cytometric assessment of myeloid cells in kidney ischemia/reperfusion injury.

    Williams, Timothy M; Wise, Andrea F; Alikhan, Maliha A; Layton, Daniel S; Ricardo, Sharon D


    Polychromatic flow cytometry is a powerful tool for assessing populations of cells in the kidney through times of homeostasis, disease and tissue remodeling. In particular, macrophages have been identified as having central roles in these three settings. However, because of the plasticity of myeloid cells it has been difficult to define a specific immunophenotype for these cells in the kidney. This study developed a gating strategy for identifying and assessing monocyte and macrophage subpopulations, along with neutrophils and epithelial cells in the healthy kidney and following ischemia/reperfusion (IR) injury in mice, using antibodies against CD45, CD11b, CD11c, Ly6C, Ly6G, F4/80, CSF-1R (CD115), MHC class II, mannose receptor (MR or CD206), an alternatively activated macrophage marker, and the epithelial cell adhesion marker (EpCAM or CD326). Backgating analysis and assessment of autofluorescence was used to extend the knowledge of various cell types and the changes that occur in the kidney at various time-points post-IR injury. In addition, the impact of enzymatic digestion of kidneys on cell surface markers and cell viability was assessed. Comparisons of kidney myeloid populations were also made with those in the spleen. These results provide a useful reference for future analyses of therapies aimed at modulating inflammation and enhancing endogenous remodeling following kidney injury.

  17. Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

    Antje eFröhling


    Full Text Available Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfil the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results.The aim of this study was to compare the inactivation effects of peracetic acid (PAA, ozonated water (O3 and cold atmospheric pressure plasma (CAPP on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s with 0.25 % PAA at 10 °C, and after treatment (10 s with 3.8 mg l-1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 min and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l-1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process

  18. Flow cytometric measurement of calcium influx in murine T cell hybrids using Fluo-3 and an organic-anion transport inhibitor.

    Baus, E; Urbain, J; Leo, O; Andris, F


    A method is described to facilitate flow cytometric analysis of calcium mobilization upon stimulation of murine T cell hybrids. In these transformed cell lines, the accuracy of cytometric measurement of free cytoplasmic calcium with Fluo-3 is compromised by the rapid loss of the intracellular dye. We have found that the addition of sulfinpyrazone, a known organic-anion transporter inhibitor in epithelial cells and in macrophages, severely impairs the leakage of the Fluo-3 probe from the cytoplasmic matrix. Under appropriate conditions, sulfinpyrazone has little effect on the cell physiology and permits the detection of calcium influx in a variety of murine T cell hybrids.

  19. Flow cytometric measurement of RNA synthesis based on bromouridine labelling and combined with measurement of DNA content or cell surface antigen

    Jensen, P O; Larsen, J; Larsen, J K


    that RNA synthesis increased within the first 24 hours of phytohemagglutinin (PHA) stimulation, reaching a maximum at 48 hours, when cells had entered the cell cycle. Using a new method for flow cytometric dual parameter analysis of BrUrd incorporation and a cell surface antigen, spontaneous RNA synthesis...




    Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by epifluoresce

  1. Flow Cytometric Measurement of [Ca2+]i and pHi in Conjugated Natural Killer Cells and K562 Target Cells during the Cytotoxic Process1,2

    van Graft, Marja; van Graft, M.; Kraan, Yvonne M.; Segers-Nolten, Gezina M.J.; Radosevic, K.; Radosevic, Katarina; de Grooth, B.G.; Greve, Jan


    We describe a flow cytometric assay that enables one to follow conjugate formation between cytotoxic cells and their target cells during the cytotoxic process. In addition, the internal calcium concentration ([Ca2+]i) and internal pH (pHi) of the conjugated cells can be monitored and directly

  2. Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

    Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A


    X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

  3. Overview of very small embryonic-like stem cells (VSELs) and methodology of their identification and isolation by flow cytometric methods.

    Zuba-Surma, Ewa K; Ratajczak, Mariusz Z


    The protocols presented here describe the procedures employed to identify and isolate very small embryonic-like stem cells (VSELs) using flow cytometric technologies including fluorescence-activated cell sorting (FACS). We describe the recommended steps in detail for their successful identification and isolation from adult tissues. These protocols were initially established to isolate such cells from murine bone marrow (BM) and human cord blood (CB) and may also be employed to isolate these primitive cells from other adult organs and embryonic tissues. Here, we focus on some critical parameters/key points required for the successful identification and purification of these rare cells by employing classical flow cytometry. In the last part of this unit, we also discuss a novel flow cytometric tool, ImageStream, an imaging flow cytometer, which allows better identification and morphological analysis of sorted cells.

  4. CD33 monoclonal antibody conjugated Au cluster nano-bioprobe for targeted flow-cytometric detection of acute myeloid leukaemia

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor, E-mail:, E-mail: [Amrita Centre for Nanoscience and Molecular Medicine, Amrita Institute of Medical Science, Cochin 682 041 (India)


    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with {approx} 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of {approx} 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in {approx} 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of {approx} 12 nm retained bright fluorescence over an extended duration of {approx} a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of {approx} 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of {approx} 8.2% in human peripheral blood cells (PBMCs) which are CD33{sup low}. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  5. A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood.

    Palmer, Clovis S; Anzinger, Joshua J; Butterfield, Tiffany R; McCune, Joseph M; Crowe, Suzanne M


    Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

  6. HLA-targeted flow cytometric sorting of blood cells allows separation of pure and viable microchimeric cell populations.

    Drabbels, Jos J M; van de Keur, Carin; Kemps, Berit M; Mulder, Arend; Scherjon, Sicco A; Claas, Frans H J; Eikmans, Michael


    Microchimerism is defined by the presence of low levels of nonhost cells in a person. We developed a reliable method for separating viable microchimeric cells from the host environment. For flow cytometric cell sorting, HLA antigens were targeted with human monoclonal HLA antibodies (mAbs). Optimal separation of microchimeric cells (present at a proportion as low as 0.01% in artificial mixtures) was obtained with 2 different HLA mAbs, one targeting the chimeric cells and the other the background cells. To verify purity of separated cell populations, flow-sorted fractions of 1000 cells were processed for DNA analysis by HLA-allele-specific and Y-chromosome-directed real-time quantitative PCR assays. After sorting, PCR signals of chimeric DNA markers in the positive fractions were significantly enhanced compared with those in the presort samples, and they were similar to those in 100% chimeric control samples. Next, we demonstrate applicability of HLA-targeted FACS sorting after pregnancy by separating chimeric maternal cells from child umbilical cord mononuclear cells. Targeting allelic differences with anti-HLA mAbs with FACS sorting allows maximal enrichment of viable microchimeric cells from a background cell population. The current methodology enables reliable microchimeric cell detection and separation in clinical specimens.

  7. Laser-based flow cytometric analysis of genotoxicity of humans exposed to ionizing radiation during the Chernobyl accident

    Jensen, Ronald H.; Bigbee, William L.; Langlois, Richard G.; Grant, Stephen G.; Pleshanov, Pavel G.; Chirkov, Andre A.; Pilinskaya, Maria A.


    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accidert at Chemobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ioniing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. This effect is similar to that which was previously observed in individuals who were exposed to ionizing radiation at Hiroshima in 1945 because of the A-bomb explosion. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure.

  8. A whole blood flow cytometric determination of platelet activation by unfractionated and low molecular weight heparin in vitro.

    Klein, Bernd; Faridi, Andreé; von Tempelhoff, G F; Heilmann, Lothar; Mittermayer, Christian; Rath, Werner


    The influence of unfractionated (Heparin-Natrium) and low-molecular heparin (Fragmin(R)) on platelet activation in whole blood was investigated by FACS analysis in vitro using antibodies against glycoprotein (gp) IIb/IIIa (CD 41), GMP 140 (CD 62P), gp 53 (CD 63) and fibrinogen. Samples were also labeled with anti-gp Ib (CD 42b). Neither unfractionated heparin (UFH) nor low molecular weight heparin (LMWH) led to significant (i.e., p<0.05) changes in fluorescence intensities of platelets labeled with anti-gp IIb/IIIa or anti-gp 53. Significant platelet activation due to unfractionated heparin could be observed by labeling with anti-GMP 140 (UFH: p=0.009; LMWH: p=0.16). The proportion of platelets with surface-bound fibrinogen was significantly increased (UFH: p=0.00006; LMWH: p=0.008). After incubation with heparins, activation ability of platelets by adenosine diphosphate (ADP) was significantly increased. The potentiating action of unfractionated heparin was larger. Therefore, flow cytometric results of platelet activation in patients receiving heparin should be interpreted carefully.

  9. Estimation of the Whitefly Bemisia tabaci Genome Size Based on k-mer and Flow Cytometric Analyses

    Wenbo Chen


    Full Text Available Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1 population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680–690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome.

  10. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Zofia Sulowska


    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  11. Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia

    Denys, B.; van der Sluijs-Gelling, A. J.; Homburg, C.; van der Schoot, C. E.; de Haas, V.; Philippe, J.; Pieters, R.; van Dongen, J. J. M.; van der Velden, V. H. J.


    Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are ob

  12. Flow cytometric measurement of RNA synthesis using bromouridine labelling and bromodeoxyuridine antibodies

    Jensen, P O; Larsen, J; Christiansen, J


    Nuclear RNA synthesis can be analysed by flow cytometry of cells labelled with 5-bromouridine (BrUrd) and stained with anti-bromodeoxyuridine (BrdUrd) antibody and FITC-conjugated secondary antibody. A panel of 5 different commercially available anti-BrdUrd antibodies was tested on cells of a HL-...

  13. Microbial Eco-Physiology of the human intestinal tract: a flow cytometric approach

    Amor, Ben K.


    This thesis describes a multifaceted approach to further enhance our view of the complex human intestinal microbial ecosystem. This approach combines me advantages of flow cyrometry (FCM), a single cell and high-throughput technology, and molecular techniques that have proven themselves to be invalu

  14. Microbial Eco-Physiology of the human intestinal tract: a flow cytometric approach

    Amor, Ben K.


    This thesis describes a multifaceted approach to further enhance our view of the complex human intestinal microbial ecosystem. This approach combines me advantages of flow cyrometry (FCM), a single cell and high-throughput technology, and molecular techniques that have proven themselves to be

  15. Microbial Eco-Physiology of the human intestinal tract: a flow cytometric approach

    Amor, Ben K.


    This thesis describes a multifaceted approach to further enhance our view of the complex human intestinal microbial ecosystem. This approach combines me advantages of flow cyrometry (FCM), a single cell and high-throughput technology, and molecular techniques that have proven themselves to be invalu

  16. Comparison of monoclonal antibodies reactive with lymphocyte subsets in routinely fixed paraffin-embedded material: flow cytometric analyses, immunoperoxidase staining and influence of fixatives.



    Full Text Available We have attempted to clarify the characteristics of monoclonal antibodies (MAbs detecting lymphocyte subsets in fixed materials. We examined by means of flow cytometric technique influences of fixatives and reactivity with malignant lymphomas (MLs. Specific markers for T-cells were UCHL1 and OPD4, which reacted especially with helper/inducer T-cells. MT1 recognized almost all of T-cells from peripheral blood and tonsils, but reacted with a part of B-MLs. As for B-cell markers, L26 was the most reliable marker for B-MLs. L26 and MB1 antigens could not be detected on living cells flow cytometrically. LN1 reacted with a part of T-cells as well as B-cells, but fluorescent intensity of the former was apparently stronger than that of the latter. Although LN2 antigen was located mainly in the cytoplasm close to the nuclear membrane immunohistochemically, it could be detected on living cells flow cytometrically. LN2 positive cells belonged to B-cells in peripheral blood and tonsils. When fixed for relatively short time, B5 and buffered formalin were better for examining MAbs than non-buffered formalin and ethanol.

  17. Current International Flow Cytometric Practices for the Detection and Monitoring of Paroxysmal Nocturnal Haemoglobinuria (PNH) clones: A UK NEQAS Survey.

    Fletcher, Matthew; Whitby, Liam; Whitby, Alison; Barnett, David


    Background Paroxysmal Nocturnal Haemoglobinuria (PNH) is a rare acquired genetic disorder, with an incidence of approximately 1.3 new cases per million population per year. Evidence from the UK National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) programme suggested major discrepancies on how PNH testing is undertaken. To investigate this we surveyed laboratories in the UK NEQAS LI PNH programme and report here the findings. Method A questionnaire was distributed to all centres registered in UK NEQAS LI flow cytometry programmes (n=1587). Comprising several subsections, it covered the majority of clinical flow cytometric practices. Participants completed a general section and then the subsections relevant to their laboratory repertoire. One subsection contained 34 questions regarding practices in PNH clone detection. Results A total of 105 laboratories returned results for the PNH section; the results demonstrated lack of consensus in all areas of PNH testing. Variation was seen in gating and testing strategies, sensitivity levels and final reporting of test results. Several incorrect practices were highlighted such as inappropriate antibody selection and failure to wash the red blood cells (RBCs) prior to analysis. Conclusion Despite the availability of consensus guidelines there appears to be no agreement in the detection and monitoring of PNH. We found only fourteen centres using methods compatible with the International Clinical Cytometry Society guidelines. Of specific note we found that no two laboratories used the same method. This technical variation could lead to incorrect diagnoses, highlighting the need for better adoption and understanding of consensus practices. This article is protected by copyright. All rights reserved.

  18. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David


    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  19. Flow cytometric determination of osmotic behaviour of animal erythrocytes toward their engineering for drug delivery

    Kostić Ivana T.


    Full Text Available Despite the fact that the methods based on the osmotic properties of the cells are the most widely used for loading of drugs in human and animal erythrocytes, data related to the osmotic properties of erythrocytes derived from animal blood are scarce. This work was performed with an aim to investigate the possibility of use the flow cytometry as a tool for determination the osmotic behaviour of porcine and bovine erythrocytes, and thus facilitate the engineering of erythrocytes from animal blood to be drug carriers. The method of flow cytometry successfully provided the information about bovine and porcine erythrocyte osmotic fragility, and made the initial steps in assessment of erythrocyte shape in a large number of erythrocytes. Although this method is not able to confirm the swelling of pig erythrocytes, it indicated to the differences in pig erythrocytes that had basic hematological parameters inside and outside the reference values. In order to apply/use the porcine and bovine erythrocytes as drug carriers, the method of flow cytometry, confirming the presence of osmotically different fractions of red blood cells, indicated that various amounts of the encapsulated drug in porcine and bovine erythrocytes can be expected.

  20. Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10.

    Süring, Katrin; Bach, Sabine; Höflich, Conny; Straff, Wolfgang


    Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 µm in diameter in inhalable particulate matter (PM10, particle size ≤10 µm in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions. So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles >0.5 µm.

  1. Flow cytometric assessment of Streptococcus mutans viability after exposure to blue light-activated curcumin.

    Manoil, Daniel; Filieri, Anna; Gameiro, Cécile; Lange, Norbert; Schrenzel, Jacques; Wataha, John C; Bouillaguet, Serge


    Streptococcus mutans biofilms are considered as primary causative agents of dental caries. Photodynamic antimicrobial chemotherapy (PACT) has been recently proposed as a strategy for inactivating dental biofilms. This study aimed to investigate the effect of blue light-activated curcumin on S. mutans viability and to explore its potential as a new anti-caries therapeutic agent. The effect of different concentrations and incubation times of photo-activated curcumin on the survival of S. mutans in planktonic and biofilm models of growth was assessed by flow cytometry. Streptococcus mutans in planktonic suspensions or biofilms formed on hydroxyapatite disks were incubated for 5 or 10min with curcumin prior to blue light activation. Bacteria were labeled with SYTO 9 and propidium iodide before viability was assessed by flow cytometry. Results were statistically analyzed using one-way ANOVA and Tukey multiple comparison intervals (α=0.05). For planktonic cultures, 0.2μM of light-activated curcumin significantly reduced S. mutans viability (p<0.05). For biofilm cultures, light-activated curcumin at concentration of 40-60μM only suppressed viability by 50% (p<0.05). Independently of the mode of growth, incubation time has no significant effect on PACT efficiency. This study indicates that blue light-activated curcumin can efficiently inactivate planktonic cultures of S. mutans whereas biofilms were more resistant to treatment. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to replicate and grow after cell sorting. Further studies seem warranted to optimize the efficacy of light-activated curcumin against S. mutans biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843).

    Filipiak, Marta; Tylko, Grzegorz; Kilarski, Wincenty


    The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak's erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm(2), respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes.

  3. Flow cytometric analysis of T lymphocyte proliferation in vivo by EdU incorporation.

    Sun, Xiaojing; Zhang, Chunpan; Jin, Hua; Sun, Guangyong; Tian, Yue; Shi, Wen; Zhang, Dong


    Monitoring T lymphocyte proliferation, especially in vivo, is essential for the evaluation of adaptive immune reactions. Flow cytometry-based proliferation assays have advantages in measuring cell division of different T lymphocyte subsets at the same time by multicolor labelling. In this study, we aimed to establish the use of 5-Ethynyl-2'-deoxyuridine (EdU) incorporation in vivo to monitor T lymphocyte proliferation by flow cytometry with an adoptive transfer model. We found that fixation followed by permeabilization preserved T cell surface antigens and had no obvious effects on the fluorescence intensity of APC, PE, PE-Cy7, FITC and PerCP-Cy5.5 when the concentration of the permeabilization reagents was optimized. However, the click reaction resulted in a significant decrease in the fluorescence intensity of PE and PE-Cy7, and surface staining after the click reaction improved the fluorescence intensity. Thus, an extra step of blocking with PBS with 3% FBS between the click reaction and cell surface staining is needed. Furthermore, the percentage of EdU-positive cells increased in a dose-dependent manner, and the saturated dose of EdU was 20mg/kg. Intraperitoneal and intravenous injection had no differences in lymphocyte proliferation detection with EdU in vivo. In addition, T cell proliferation measured by EdU incorporation was comparable to BrdU but was lower than CFSE labelling. In conclusion, we optimized the protocols for EdU administration in vivo and staining in vitro, providing a feasible method for the measurement of T lymphocyte proliferation with EdU incorporation by flow cytometry in vivo.

  4. Quantitative assessment of BAX transcript and flow cytometric expression in acute myeloid leukemia: a prospective study.

    Sharawat, Surender Kumar; Raina, Vinod; Kumar, Lalit; Sharma, Atul; Bakhshi, Radhika; Vishnubhatla, Sreenivas; Gupta, Ritu; Bakhshi, Sameer


    Quantitative assessment of BAX transcripts and protein in acute myeloid leukemia (AML). We quantitatively evaluated BAX gene transcripts by real-time polymerase chain reaction (TaqMan probe chemistry) and protein expression by flow cytometry. Consecutive 112 AML patients with a median age of 16 (1-59) years were recruited in the study. By flow cytometry, the percentage expression was in linear correlation with relative median fluorescent intensity (RMFI; R = 0.4425; P BAX with its RMFI (R = -0.0559; P = 0.586). The expression of the BAX at both protein and transcript level was significantly higher in AML patients as compared with normal control. RMFI of the BAX were higher in the cohort with lower white blood cell count (P = 0.029). None of the other baseline characteristics correlated with either the BAX transcript or the RMFI. BAX expression did not correlate with complete remission rate, event free, disease free, and overall survival. BAX gene expression in AML was evaluated first time with two different methods but did not correlate with the survival outcome.

  5. Flow cytometric measurement of the cellular propagation of TDP-43 aggregation.

    Zeineddine, Rafaa; Whiten, Daniel R; Farrawell, Natalie E; McAlary, Luke; Hanspal, Maya A; Kumita, Janet R; Wilson, Mark R; Yerbury, Justin J


    Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.

  6. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters.

    Vorobjev, Ivan A; Buchholz, Kathrin; Prabhat, Prashant; Ketman, Kenneth; Egan, Elizabeth S; Marti, Matthias; Duraisingh, Manoj T; Barteneva, Natasha S


    Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene

  7. Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters

    Vorobjev Ivan A


    Full Text Available Abstract Background Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP labelling is complicated by autofluorescence (AF of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. Methods Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. Results A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP, AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. Discussion Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis

  8. Flow cytometric applicability to evaluate UV inactivation of phytoplankton in marine water samples.

    Olsen, Ranveig Ottoey; Hess-Erga, Ole-Kristian; Larsen, Aud; Thuestad, Gunnar; Tobiesen, August; Hoell, Ingunn Alne


    Disinfection of microbes is of importance to prevent the spread of pathogens and non-indigenous species in the environment. Here we test the applicability of using flow cytometry (FCM) to evaluate inactivation of the phytoplankter Tetraselmis suecica after UV irradiation and labeling with the esterase substrate 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). Non-irradiated and UV irradiated samples were analyzed with the plate count technique and FCM for 24 days. The numbers of colony forming units were used as a standard to develop a FCM protocol. Our protocol readily distinguishes live and dead cells, but challenges were encountered when determining whether UV damaged cells are dying or repairable. As damaged cells can represent a risk to aquatic organisms and/or humans, this was taken into account when developing the FCM protocol. In spite of the above mentioned challenges we argue that FCM represents an accurate and rapid method to analyze T. suecica samples.

  9. Determination of micro-litre volumes with high accuracy for flow cytometric blood cell counting

    Reitz, S.; Kummrow, A.; Kammel, M.; Neukammer, J.


    We have gravimetrically calibrated the volumes dispensed by 1 mL syringes in the range between 1 µL and 100 µL using ultra-pure water. Protocols are based on series of consecutive difference measurements of masses in order to precisely compensate for evaporation, being the most important disturbing quantity. We determined expanded uncertainties of volume measurements for glass syringes of typically 0.2% (expansion factor 2) when dispensing volumes of 10 µL. For polypropylene syringes, selected with respect to the manufacturer, expanded uncertainties of 0.25% (expansion factor 2) were observed. Calibrated syringes were applied for measuring concentrations of blood cells in a flow cytometer demonstrating the capability to determine reference measurement values. Since the direct interaction of blood cells and syringe walls may lead to cell adhesion, glass syringes as well as (disposable) polypropylene syringes were calibrated.

  10. Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells

    Møller-Larsen, A; Brudek, T; Petersen, T


    as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted......Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable...... assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients...

  11. Flow cytometric analysis of lymphocytes and lymphocyte subpopulations in induced sputum from patients with asthma

    Yutaro Shiota


    Full Text Available Study objectives were to compare the numbers of lymphocytes and lymphocyte subpopulations in induced sputum from asthmatic patients and from healthy subjects, and to determine the effect of inhaled anti-asthmatic steroid therapy on these cell numbers. Hypertonic saline inhalation was used to non-invasively induce sputum samples in 34 patients with bronchial asthma and 21 healthy subjects. The sputum samples were reduced with dithioerythritol and absolute numbers of lymphocytes and lymphocyte subpopulations were assessed by direct immunofluorescence and flow cytometry. To assess the effect of beclomethasone dipropionate (BDP on induced sputum, numbers of lymphocytes and lymphocyte subpopulations in sputum also were evaluated after 4 weeks of BDP inhalation treatment in seven asthmatic patients. An adequate sample was obtained in 85.3% of patients with asthma and in 79.2% of the healthy subjects. Induced sputum from patients with asthma had increased numbers of lymphocytes (P = 0.009; CD4+ cells (P = 0.044; CD4+ cells-bearing interleukin-2 receptor (CD25; P = 0.016; and CD4+ cells bearing human histocompatibility leukocyte antigen (HLA-DR (P = 0.033. CD8+ cells were not increased in asthmatic patients. In patients treated with inhaled steroids, numbers of lymphocytes, CD4+ cells, CD25-bearing CD4+ cells and HLA-DR-bearing CD4+ cells in sputum decreased from pretreatment numbers (P = 0.016, 0.002, 0.003 and 0.002, respectively. Analysis of lymphocytes in induced sputum by flow cytometry is useful in assessing bronchial inflammation, and activated CD4+ lymphocytes may play a key role in the pathogenesis of airway inflammation in bronchial asthma.

  12. Assessment of a five-color flow cytometric assay for verifying automated white blood cell differentials

    HUANG Chun-mei; YU Lian-hui; PU Cheng-wei; WANG Xin; WANG Geng; SHEN Li-song; WANG Jian-zhong


    Background White blood cell (WBC) counts and differentials performed using an automated cell counter typically require manual microscopic review.However,this last step is time consuming and requires experienced personnel.We evaluated the clinical efficiency of using flow cytometry (FCM) employing a six-antibody/five-color reagent for verifying automated WBC differentials.Methods A total of 56 apparently healthy samples were assessed using a five-color flow cytometer to verify the normal reference ranges of WBC differentials.WBC differentials of 622 samples were also determined using both a cell counter and FCM.These results were then confirmed using manual microscopic methods.Results The probabilities for all of the parameters of WBC differentials exceeded the corresponding normal reference ranges by no more than 7.5%.The resulting WBC differentials were well correlated between FCM and the cell counter (r >0.88,P <0.001),except in the case of basophils.Neutrophils,lymphocytes,and eosinophils were well correlated between FCM and standard microscopic cytology assessment (r >0.80,P <0.001).The sensitivities of FCM for identification of immature granulocytes and blast cells (72.03% and 22.22%,respectively) were higher than those of the cell counter method (44.92% and 11.11%,respectively).The specificities of FCM were all above 85%,substantially better than those of the cell counter method.Conclusion These five-color FCM assays could be applied to accurately verify abnormal results of automated assessment of WBC differentials.

  13. Flow cytometric assessment of microbial abundance in the near-field area of seawater reverse osmosis concentrate discharge

    Van Der Merwe, Riaan


    The discharge of concentrate and other process waters from seawater reverse osmosis (SWRO) plant operations into the marine environment may adversely affect water quality in the near-field area surrounding the outfall. The main concerns are the increase in salt concentration in receiving waters, which results in a density increase and potential water stratification near the outfall, and possible increases in turbidity, e.g., due to the discharge of filter backwash waters. Changes in ambient water quality may affect microbial abundance in the area, for example by hindering the photosynthesis process or disrupting biogenesis. It is widely accepted that marine biodiversity is lower in more extreme conditions, such as high salinity environments. As aquatic microbial communities respond very rapidly to changes in their environment, they can be used as indicators for monitoring ambient water quality. The objective of this study was to assess possible changes in microbial abundance as a result of concentrate discharge into the near-field area (<. 25. m) surrounding the outfall of the King Abdullah University of Science and Technology (KAUST) SWRO plant. Flow cytometric (FCM) analysis was conducted in order to rapidly determine microbial abundance on a single-cell level in 107 samples, taken by diving, from the discharge area, the intake area and two control sites. FCM analysis combined the measurement of distinct scatter of cells and particles, autofluorescence of cyanobacteria and algae, and fluorescence after staining of nucleic acids with SYBR® Green for a total bacterial count. The results indicate that changes in microbial abundance in the near-field area of the KAUST SWRO outfall are minor and appear to be the result of a dilution effect rather than a direct impact of the concentrate discharge. © 2014 Elsevier B.V.

  14. Histopathologic and Flow-Cytometric Analysis of Neoplastic and Benign “background” Tissue in Breast Carcinoma Resections

    Daniel W. Visscher


    Full Text Available Two-color, multiparametric synthesis phase fraction (SPF analysis of cytokeratin-labeled epithelial cells was flow cytometrically performed on both benign (SPFb and malignant tissue samples (if available, SPFt from 132 mastectomy/lumpectomy specimens. These data were then correlated with clinicopathologic features, including (1 tumor differentiation, (2 the proportion of tumor comprised of duct carcinoma-in situ (DCIS, and (3 the histology of accompanying benign breast tissue, classified by predominant microscopic pattern as intact, normal terminal duct lobular units (NTDLU, 34% of cases, atrophic (AT, 33% of cases, proliferative fibrocystic (PFC, 26% of cases, and non-proliferative fibrocystic (NPFC, 7% of cases. SPFt was inversely correlated with extent of DCIS (DCIS =0 – 20% tumor volume – 12.7% mean SPFt, vs. DCIS >20% tumor volume – 6.4% mean SPFt, p = 0.001. SPFt also correlated with the histology of background benign breast tissue (NTDLU – 14.8% mean SPFt vs. AT – 6.9% mean SPFt vs. PFC – 12.7% mean SPFt, p = 0.05 but it did not correlate with patient age or SPFb (overall mean =0.73%. SPFb was correlated with patient age (>56 yr – 0.59% mean SPFb vs. < yr – 0.84% mean SPFb, p = 0.02, with background histology (NTDLU – 1.1% mean SPFb vs. AT – 0.43% mean SPFb vs. PFC – 0.70% mean SPFb, p < 0.02 and with the grade of the neoplasm (well/moderate – 0.58% mean vs. poorly differentiated – 0.85% mean, p = 0.04. Patients having a background of PFC were significantly older than patients with a background of NTDLU (45.2 yr vs. 60.2 yr, p = 0.01.

  15. Performance of the flow cytometric E-screen assay in screening estrogenicity of pure compounds and environmental samples

    Vanparys, Caroline, E-mail: [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Depiereux, Sophie; Nadzialek, Stephanie [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); Robbens, Johan; Blust, Ronny [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); Kestemont, Patrick [Research Unit in Organismal Biology (URBO), University of Namur (FUNDP), Namur (Belgium); De Coen, Wim [Laboratory of Ecophysiology, Biochemistry and Toxicology, University of Antwerp, Antwerp (Belgium); European Chemicals Agency (ECHA), Helsinki (Finland)


    In vitro estrogenicity screens are believed to provide a first prioritization step in hazard characterization of endocrine disrupting chemicals. When applied to complex environmental matrices or mixture samples, they have been indicated valuable in estimating the overall estrogen-mimicking load. In this study, the performance of an adapted format of the classical E-screen or MCF-7 cell proliferation assay was profoundly evaluated to rank pure compounds as well as influents and effluents of sewage treatment plants (STPs) according to estrogenic activity. In this adapted format, flow cytometric cell cycle analysis was used to allow evaluation of the MCF-7 cell proliferative effects after only 24 h of exposure. With an average EC{sub 50} value of 2 pM and CV of 22%, this assay appears as a sensitive and reproducible system for evaluation of estrogenic activity. Moreover, estrogenic responses of 17 pure compounds corresponded well, qualitatively and quantitatively, with other in vitro and in vivo estrogenicity screens, such as the classical E-screen (R{sup 2} = 0.98), the estrogen receptor (ER) binding (R{sup 2} = 0.84) and the ER transcription activation assay (R{sup 2} = 0.87). To evaluate the applicability of this assay for complex samples, influents and effluents of 10 STPs covering different treatment processes, were compared and ranked according to estrogenic removal efficiencies. Activated sludge treatment with phosphorus and nitrogen removal appeared most effective in eliminating estrogenic activity, followed by activated sludge, lagoon and filter bed. This is well in agreement with previous findings based on chemical analysis or biological activity screens. Moreover, ER blocking experiments indicated that cell proliferative responses were mainly ER mediated, illustrating that the complexity of the end point, cell proliferation, compared to other ER screens, does not hamper the interpretation of the results. Therefore, this study, among other E-screen studies

  16. Evaluation of Prognostic Factors Following Flow-Cytometric DNA Analysis after Cytokeratin Labelling: II. Cervical and Endometrial Cancer

    Pauline Wimberger


    Full Text Available In gynecologic oncology valid prognostic factors are necessary to define biologically similar subgroups for analysis of therapeutic efficacy. This study is the first published prospective study concerning prognostic significance of DNA ploidy and S‐phase fraction in cervical and endometrial cancer following enrichment of tumor cells by cytokeratin labelling. Epithelial cells were labeled by FITC‐conjugated cytokeratin antibody (CK 5, 6, 8, and CK 17 prior to flow cytometric cell cycle analysis in 91 specimens of cervical cancer and 73 samples of endometrial cancer. In cervical cancer neither DNA‐ploidy nor S‐phase fraction were relevant prognostic parameters. But CV of the G0G1‐peak showed prognostic relevance in cervical cancer cells, even in multivariate analysis. This interesting observation, however, seems to have no therapeutic consequence due to the small discrimination capacity of CV. In endometrial carcinoma, gross DNA‐aneuploidy (DNA‐index > 1.3 and a high percentage of proliferating cells (>75th percentile were univariate and multivariate highly significant prognostic factors for recurrence‐free survival. Especially DNA‐aneuploidy (DI>1.3 is one of the most important independent molecular biological prognostic factors. While diagnostic curettage we could identify risk patients even preoperatively by determination of the prognostic factors like histologic tumor type, grading, cervical involvement and DNA‐ploidy. Thereby these patients could be treated primarily in an oncologic center. In conclusion, our investigations showed that the determination of DNA‐ploidy should be done in endometrial carcinoma. In cervical cancer no clinical significance for determination of DNA‐parameters was found.

  17. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.

    Sun, Shuang; Yang, Xiao; Wang, Haifeng; Zhao, Yun; Lin, Yan; Ye, Chen; Fang, Xiangdong; Hang, Haiying


    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.

  18. Relevance of Flow Cytometric Auto-Crossmatch to the Post-transplant Course of Kidney Transplant Recipients.

    Demir, E; Yeğit, O; Erol, A; Akgül, S U; Çalışkan, B; Bayraktar, A; Çalışkan, Y; Türkmen, A; Savran, F O; Sever, M S


    The crossmatch test is essential prior to kidney transplantation (tx) to confirm compatibility between the donor and the recipient. However, its results can be misleading due to "undetectable antibodies" in the recipient's serum. To establish if undetectable autoantibodies are responsible for a positive result, an auto-crossmatch test can be performed. In this study, we aim to determine the long-term prognostic value of auto-flow cytometric auto-crossmatch (FCXM) test on kidney survival in kidney tx recipients. The primary outcome variable was reduced renal function. Secondary endpoints were incidence of biopsy-confirmed chronic antibody-mediated rejection (CAMR) and recurrent glomerulonephritis (GN). There were no differences regarding initial serum creatinine levels between the study and control groups (P = .441). Patients who had positive auto-B FCXM had a significantly reduced renal function compared with the control group (P = .016). Four patients developed biopsy-confirmed CAMR in the study group and 1 patient in the control group (P = .047). Five patients had biopsy-confirmed recurrent GN in the GN study group, and only 1 patient had recurrent GN in the GN control group (P = .026). Kidney transplant recipients with positive auto-FCXM test had significantly reduced renal function and a higher incidence of recurrent GN and CAMR compared with the control group. The findings of this study suggest a potential role of auto-antibody causing positive auto-FCXM test result, meanwhile increasing the risk of CAMR, recurrent GN, and new-onset diabetes after tx. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.

    Koopman, G; Reutelingsperger, C P; Kuijten, G A; Keehnen, R M; Pals, S T; van Oers, M H


    Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.

  20. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring.

    Van Nevel, S; Koetzsch, S; Proctor, C R; Besmer, M D; Prest, E I; Vrouwenvelder, J S; Knezev, A; Boon, N; Hammes, F


    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  1. Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios.

    Novak, E J; Rabinovitch, P S


    Measurement of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo-1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo-3. Both Fura Red and fluo-3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo-3/Fura Red ratio is greater than that achieved with indo-1, while the intercellular variation in measurement is similar to that seen with indo-1. Analyses can be simultaneously combined with immunofluorescent detection of PE-labeled antibodies to enable [Ca2+]i measurement within cell subsets.

  2. Neuropathological similarities and differences between schizophrenia and bipolar disorder: a flow cytometric postmortem brain study.

    Yoshitaka Hayashi

    Full Text Available Recent studies suggest that schizophrenia (SCH and bipolar disorder (BPD may share a similar etiopathology. However, their precise neuropathological natures have rarely been characterized in a comprehensive and quantitative fashion. We have recently developed a rapid, quantitative cell-counting method for frozen unfixed postmortem brains using a flow cytometer. In the present study, we not only counted stained nuclei, but also measured their sizes in the gray matter of frontopolar cortices (FPCs and inferior temporal cortices (ITCs from patients with SCH or BPD, as well as in that from normal controls. In terms of NeuN(+ neuronal nuclei size, particularly in the reduced densities of small NeuN(+ nuclei, we found abnormal distributions present in the ITC gray matter of both patient groups. These same abnormalities were also found in the FPCs of SCH patients, whereas in the FPCs of BPD patients, a reduction in oligodendrocyte lineage (olig2(+ cells was much more common. Surprisingly, in the SCH FPC, normal left-greater-than-right asymmetry in neural nuclei densities was almost completely reversed. In the BPD FPC, this asymmetry, though not obvious, differed significantly from that in the SCH FPC. These findings indicate that while similar neuropathological abnormalities are shared by patients with SCH or BPD, differences also exist, mainly in the FPC, which may at least partially explain the differences observed in many aspects in these disorders.

  3. Evaluation of chromatin condensation in human spermatozoa: a flow cytometric assay using acridine orange staining.

    Golan, R; Shochat, L; Weissenberg, R; Soffer, Y; Marcus, Z; Oschry, Y; Lewin, L M


    The quality of sperm chromatin is an important factor in fertilization and is especially critical where one spermatozoon is artificially selected for fertilizing an egg (as in intracytoplasmic sperm injection). In this study, flow cytometry after staining of human spermatozoa with Acridine Orange was used to study chromatin structure. A method is described for estimating the percentage of cells in a human sperm sample that have completed epididymal maturation in regard to chromatin condensation. Of the 121 samples of the semen that were examined, nine contained a higher percentage of hypocondensed spermatozoa and six samples contained elevated amounts of hypercondensed spermatozoa. In addition to aberrancies in chromatin condensation other defects showed up as satellite populations of spermatozoa with higher than normal ratios of red/green fluorescence after Acridine Orange staining. Such defects were found in 15 semen samples. The use of swim-up and Percoll gradient centrifugation methods was shown to improve the percentage of spermatozoa with normal chromatin structure in some samples with poor initial quality.

  4. Development of a novel flow cytometric approach to evaluate fish sperm chromatin using fixed samples

    Jenkins, Jill A.


    The integrity of the paternal DNA is essential for the accurate transmission of genetic information, yet fertilization is not inhibited by chromatin breakage. Some methods are available for the sensitive detection of DNA damage and can be applied in studies of environmental toxicology, carcinogenesis, aging, and assisted reproduction techniques in both clinical and experimental settings. Because semen samples obtained from remote locations undergo chromatin damage prior to laboratory assessment, the present study was undertaken to evaluate treatments for effective chromatin staining in the development of a DNA fragmentation assay using fixed milt from yellow perch (Perca flavescens). Similar to the sperm chromatin structure assay (SCSA), susceptibility of nuclear DNA to acid-induced denaturation was measured by flow cytometry (FCM). Use of 10% buffered formalin for milt fixation allowed easier peak discrimination than 4% paraformaldehyde. The effects of time and temperature of incubation in 0.08 N HCl were evaluated in order to determine the ideal conditions for promoting DNA decondensation and making strand breaks more available for staining and detection by FCM. The best results were obtained with incubation at 37°C for 1 minute, followed by cold propidium iodide staining for 30 minutes.

  5. Flow cytometric functional analysis of multidrug resistance by Fluo-3: a comparison with rhodamine-123.

    Koizumi, S; Konishi, M; Ichihara, T; Wada, H; Matsukawa, H; Goi, K; Mizutani, S


    Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression.

  6. Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method

    Prest, Emmanuelle I E C


    Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing. © 2013 Elsevier Ltd.

  7. Flow Cytometric Analysis of Leishmania Reactive CD4+/CD8+ Lymphocyte Proliferation in Cutaneous Leishmaniasis

    H Keshavarz


    Full Text Available Background: Determination of the division history of T cells in vitro is helpful in the study of effector mechanisms against infections. Technique described here uses the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE to monitor the proliferation. Methods: In a cross sectional study, blood samples were collected from 7 volunteers with history of cutaneous leishmania­sis (CL and one healthy control from endemic areas in Isfahan province who referred to the Center for Research and Training in Skin Diseases and Leprosy (CRTSDL, then CD4+/CD8+ lymphocytes and CD14+ monocytes were isolated from peri­pheral blood mononuclear cells (PBMC using mAbs and magnetic nanoparticles. CFSE labeled CD4+ or CD8+ lympho­cytes cultured with autologous monocytes in the presence of PHA, SLA, live Leishmania major or as control with­out sti­mulation. Cells were harvested after 7 days and were analyzed using flow cytometry. Results: Five consecutive divisions were monitored separately. Stimulation of CD4+ or CD8+ lymphocytes from CL sub­jects with SLA showed a significant difference in proliferation comparing with unstimulated cells (P< 0.05. The signifi­cant difference in the percentages of CD4+ cells stimulated with SLA was revealed at different divisions for each subject. In CD8+ lymphocyte, significant stronger stimulation of SLA was evident later in the proliferation process. The mean number of divisions in both CD4+/CD8+ lymphocytes stimulated with SLA was significantly greater than when stimulated with live L. major (P=0.007 / P=0.012, respectively Conclusion: The percentage of divided cells might be calculated separately in each division. The cells remained active following CFSE staining and there is possibility of functional analysis simultaneously.

  8. Monitoring microbiological changes in drinking water systems using a fast and reproducible flow cytometric method.

    Prest, E I; Hammes, F; Kötzsch, S; van Loosdrecht, M C M; Vrouwenvelder, J S


    Flow cytometry (FCM) is a rapid, cultivation-independent tool to assess and evaluate bacteriological quality and biological stability of water. Here we demonstrate that a stringent, reproducible staining protocol combined with fixed FCM operational and gating settings is essential for reliable quantification of bacteria and detection of changes in aquatic bacterial communities. Triplicate measurements of diverse water samples with this protocol typically showed relative standard deviation values and 95% confidence interval values below 2.5% on all the main FCM parameters. We propose a straightforward and instrument-independent method for the characterization of water samples based on the combination of bacterial cell concentration and fluorescence distribution. Analysis of the fluorescence distribution (or so-called fluorescence fingerprint) was accomplished firstly through a direct comparison of the raw FCM data and subsequently simplified by quantifying the percentage of large and brightly fluorescent high nucleic acid (HNA) content bacteria in each sample. Our approach enables fast differentiation of dissimilar bacterial communities (less than 15 min from sampling to final result), and allows accurate detection of even small changes in aquatic environments (detection above 3% change). Demonstrative studies on (a) indigenous bacterial growth in water, (b) contamination of drinking water with wastewater, (c) household drinking water stagnation and (d) mixing of two drinking water types, univocally showed that this FCM approach enables detection and quantification of relevant bacterial water quality changes with high sensitivity. This approach has the potential to be used as a new tool for application in the drinking water field, e.g. for rapid screening of the microbial water quality and stability during water treatment and distribution in networks and premise plumbing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. A numerical analysis model for interpretation of flow cytometric studies of ex vivo phagocytosis.

    Ted S Strom

    Full Text Available The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m, the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha, and the probability of phagocytosis per adsorbed target (p. The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+ and [adsorption(-, phagocytosis(+] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.

  10. Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring

    Van Nevel, S.


    Drinking water utilities and researchers continue to rely on the century-old heterotrophic plate counts (HPC) method for routine assessment of general microbiological water quality. Bacterial cell counting with flow cytometry (FCM) is one of a number of alternative methods that challenge this status quo and provide an opportunity for improved water quality monitoring. After more than a decade of application in drinking water research, FCM methodology is optimised and established for routine application, supported by a considerable amount of data from multiple full-scale studies. Bacterial cell concentrations obtained by FCM enable quantification of the entire bacterial community instead of the minute fraction of cultivable bacteria detected with HPC (typically < 1% of all bacteria). FCM measurements are reproducible with relative standard deviations below 3% and can be available within 15 min of samples arriving in the laboratory. High throughput sample processing and complete automation are feasible and FCM analysis is arguably less expensive than HPC when measuring more than 15 water samples per day, depending on the laboratory and selected staining procedure(s). Moreover, many studies have shown FCM total (TCC) and intact (ICC) cell concentrations to be reliable and robust process variables, responsive to changes in the bacterial abundance and relevant for characterising and monitoring drinking water treatment and distribution systems. The purpose of this critical review is to initiate a constructive discussion on whether FCM could replace HPC in routine water quality monitoring. We argue that FCM provides a faster, more descriptive and more representative quantification of bacterial abundance in drinking water.

  11. Flow cytometric sorting of fecal bacteria after in situ hybridization with polynucleotide probes.

    Bruder, Lena M; Dörkes, Marcel; Fuchs, Bernhard M; Ludwig, Wolfgang; Liebl, Wolfgang


    The gut microbiome represents a key contributor to human physiology, metabolism, immune function, and nutrition. Elucidating the composition and genetics of the gut microbiota under various conditions is essential to understand how microbes function individually and as a community. Metagenomic analyses are increasingly used to study intestinal microbiota. However, for certain scientific questions it is sufficient to examine taxon-specific submetagenomes, covering selected bacterial genera in a targeted manner. Here we established a new variant of fluorescence in situ hybridization (FISH) combined with fluorescence-activated cell sorting (FACS), providing access to the genomes of specific taxa belonging to the complex community of the intestinal microbiota. In contrast to standard oligonucleotide probes, the RNA polynucleotide probe used here, which targets domain III of the 23S rRNA gene, extends the resolution power in environmental samples by increasing signal intensity. Furthermore, cells hybridized with the polynucleotide probe are not subjected to harsh pretreatments, and their genetic information remains intact. The protocol described here was tested on genus-specifically labeled cells in various samples, including complex fecal samples from different laboratory mouse types that harbor diverse intestinal microbiota. Specifically, as an example for the protocol described here, RNA polynucleotide probes could be used to label Enterococcus cells for subsequent sorting by flow cytometry. To detect and quantify enterococci in fecal samples prior to enrichment, taxon-specific PCR and qPCR detection systems have been developed. The accessibility of the genomes from taxon-specifically sorted cells for subsequent molecular analyses was demonstrated by amplification of functional genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

  12. Flow-cytometric study of vital cellular functions in Escherichia coli during solar disinfection (SODIS).

    Berney, Michael; Weilenmann, Hans-Ulrich; Egli, Thomas


    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of 80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight.

  13. Color encoded microbeads-based flow cytometric immunoassay for polycyclic aromatic hydrocarbons in food

    Meimaridou, Anastasia, E-mail: [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Haasnoot, Willem; Noteboom, Linda; Mintzas, Dimitrios [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Pulkrabova, Jana; Hajslova, Jana [Department of Food Chemistry and Analysis, Institute of Chemical Technology Prague, Technicka 3, 166 28 Prague 6 (Czech Republic); Nielen, Michel W.F. [RIKILT-Institute of Food Safety, Wageningen UR, P.O. Box 230, 6700 AE Wageningen (Netherlands); Wageningen University, Laboratory of Organic Chemistry, Dreijenplein 8, 6703 HB Wageningen (Netherlands)


    Food contamination caused by chemical hazards such as persistent organic pollutants (POPs) is a worldwide public health concern and requires continuous monitoring. The chromatography-based analysis methods for POPs are accurate and quite sensitive but they are time-consuming, laborious and expensive. Thus, there is a need for validated simplified screening tools, which are inexpensive, rapid, have automation potential and can detect multiple POPs simultaneously. In this study we developed a flow cytometry-based immunoassay (FCIA) using a color-encoded microbeads technology to detect benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) in buffer and food extracts as a starting point for the future development of rapid multiplex assays including other POPs in food, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs). A highly sensitive assay for BaP was obtained with an IC{sub 50} of 0.3 {mu}g L{sup -1} using a monoclonal antibody (Mab22F12) against BaP, similar to the IC{sub 50} of a previously described enzyme-linked immunosorbent assay (ELISA) using the same Mab. Moreover, the FCIA was 8 times more sensitive for BaP compared to a surface plasmon resonance (SPR)-based biosensor immunoassay (BIA) using the same reagents. The selectivity of the FCIAs was tested, with two Mabs against BaP for 25 other PAHs, including two hydroxyl PAH metabolites. Apart from BaP, the FCIAs can detect PAHs such as indenol[1,2,3-cd]pyrene (IP), benz[a]anthracene (BaA), and chrysene (CHR) which are also appointed by the European Food Safety Authority (EFSA) as suitable indicators of PAH contamination in food. The FCIAs results were in agreement with those obtained with gas chromatography-mass spectrometry (GC-MS) for the detection of PAHs in real food samples of smoked carp and wheat flour and has great potential for the future routine application of this assay in a simplex or multiplex format in combination with simplified extraction

  14. Flow-cytometric determination of genotoxic effects of exposure to petroleum in mink and sea otters

    Bickham, J.W.; Mazet, J.A.; Blake, J.; Smolen, M.J.; Lou, Y.; Ballachey, B.E.


    Three experiments were conducted to investigate the genotoxic effects of crude oil on mink and sea otters, In the first experiment, the effects on mink of chronic exposure to weathered Prudhoe Bay crude oil were studied, Female mink were fed a diet that included weathered crude oil for a period of 3 weeks prior to mating, during pregnancy and until weaning. Kits were exposed through lactation and by diet after weaning until 4 months of age. Kidney and liver tissues of the kits were examined using flow cytometry (FCM) and it was found that the genome size was increased in kidney samples from the experimental group compared to the control group. This effect was probably due to some type of DNA amplification and it could have been inherited from the exposed mothers or have been a somatic response to oil exposure in the pups, No evidence of clastogenic effects, as measured by the coefficient of variation (CV) of the G(1) peak, was found in kidney or liver tissue. In the second experiment, yearling female mink were exposed either by diet or externally to crude oil or bunker C fuel oil. Evidence for clastogenic damage was found in spleen tissue for the exposure groups, but not in kidney tissue. No evidence of increased genome size was observed. In the third experiment, blood was obtained from wild-caught sea otters in Prince William Sound. The sea otters represented two populations: one from western Prince William Sound that was potentially exposed to oil from the Exxon Valdez oil spill and a reference population from eastern Prince William Sound that did not receive oil from the spill. The spill had occurred 1.5 years prior to obtaining the blood samples. Although the mean CVs did not differ between the populations, the exposed population had a significantly higher variance of CV measurements and five out of 15 animals from the exposed population had CVs higher than the 95% confidence limits of the reference population, It is concluded that FCM is a sensitive indicator

  15. Flow Cytometric Quantification of Peripheral Blood Cell β-Adrenergic Receptor Density and Urinary Endothelial Cell-Derived Microparticles in Pulmonary Arterial Hypertension.

    Jonathan A Rose

    Full Text Available Pulmonary arterial hypertension (PAH is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH.

  16. The effects of orange juice clarification on the physiology of Escherichia coli; growth-based and flow cytometric analysis.

    Anvarian, Amir H P; Smith, Madeleine P; Overton, Tim W


    Orange juice (OJ) is a food product available in various forms which can be processed to a greater or lesser extent. Minimally-processed OJ has a high consumer perception but presents a potential microbiological risk due to acid-tolerant bacteria. Clarification of OJ (such as removal of cloud) is a common processing step in many OJ products. However, many of the antimicrobial components of OJ such as essential oils are present in the cloud fraction. Here, the effect of clarification by filtration on the viability and physiology of Escherichia coli K-12 was tested using total viable count (TVC) and flow cytometric (FCM) analysis. The latter technique was also used to monitor intracellular pH during incubation in OJ. Removal of the OJ cloud fraction was shown to have dramatic effects on bacterial viability and physiology during storage at a range of incubation temperatures. For instance, at 4 °C, a significantly lower number of healthy cells and a significantly higher number of injured cells were observed in 0.22 μm-filtered OJ at 24h post-inoculation, compared to filtered OJ samples containing particles between 0.22 μm and 11 μm in size. Similarly, there was a significant difference between the number of healthy bacteria in the 0.7 μm-filtered OJ and both 0.22 μm-filtered and 1.2 μm-filtered OJ after 24 hour incubation at 22.5 °C. This indicated that OJ cloud between 0.7 μm and 0.22 μm in size might have an adverse effect on the viability of E. coli K-12. Furthermore, FCM allowed the rapid analysis of bacterial physiology without the requirement for growth on agar plates, and revealed the extent of the viable but non-culturable (VBNC) population. For example, at 4 °C, while the FCM viable count did not substantially decrease until 48 h, decreases in TVC were observed between 0 and 48 hour incubation, due to a subset of injured bacteria entering the VBNC state, hence being unable to grow on agar plates. This study highlights the application of FCM in

  17. Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: protocol optimization and transferability assessment.

    Bryce, Steven M; Avlasevich, Svetlana L; Bemis, Jeffrey C; Tate, Matthew; Walmsley, Richard M; Saad, Frédéric; Van Dijck, Kris; De Boeck, Marlies; Van Goethem, Freddy; Lukamowicz-Rajska, Magdalena; Elhajouji, Azeddine; Dertinger, Stephen D


    An automated approach for scoring in vitro micronuclei (MN) has been described in which flow cytometric analysis is combined with compound exposure, processing, and sampling in a single 96-well plate (Bryce SM et al. [2010]: Mutat Res 703:191-199). The current report describes protocol optimization and an interlaboratory assessment of the assay's transferability and reproducibility. In a training phase, the methodology was refined and collaborating laboratories were qualified by repeatedly testing three compounds. Second, a set of 32 chemicals comprised of reference genotoxicants and presumed non-genotoxicants was tested at each of four sites. TK6 cells were exposed to 10 closely spaced compound concentrations for 1.5- to 2-cell population doublings, and were then stained and lysed for flow cytometric analysis. MN frequencies were determined by evaluating ≥ 5,000 cells per replicate well, and several indices of cytotoxicity were acquired. The prevalence of positive results varied according to the MN-fold increase used to signify a genotoxic result, as well as the endpoint used to define a cytotoxicity limit. By varying these parameters, assay sensitivity and specificity values ranged from 82 to 98%, and 86 to 97%, respectively. In a third phase, one laboratory tested a further six genotoxicants and five non-genotoxic apoptosis inducers. In these experiments assay specificity was markedly improved when top concentration selection was based on two cytotoxicity endpoints-relative survival and quantification of ethidium monoazide-positive events. Collectively, the results indicate that the miniaturized assay is transferable across laboratories. The 96-well format consumes considerably less compound than conventional in vitro MN test methods, and the high information content provided by flow cytometry helps guard against irrelevant positive results arising from overt toxicity.

  18. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Angela Buys


    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  19. Flow cytometric method for in situ preparation of standard materials of a small defined number of microbial cells with colony-forming potentiality.

    Matsuoka, Hideaki; Nakano, Koichiro; Takatani, Norimasa; Yoshida, Tomonori; Igimi, Shizunobu; Saito, Mikako


    Standard materials of a small defined number of cells with colony-forming potentiality are essential for the rational validation of food microbiological methods. An in situ flow cytometric method using viable staining with 6-carboxyfluorescein diacetate (CFDA) and tryptic soy agar (TSA) was previously proposed and its feasibility was demonstrated with five strains. In this study, this method was applied to 16 strains to support its broad applicability. The cell sorting gate was previously determined based on the CFDA stainability alone. Now the structural properties of cells designated by forward and side-scattering intensities have been introduced as the second gating criteria. Under the optimum gate condition, 100 cells have been selected and sorted on TSA. Consequently, a 95% or higher colony-forming rate has been attained for every strain. A successful application to microaerophilic Campylobacter spp. is especially of great importance because it suggests further broader applicability.

  20. Fluorescence in situ hybridization and sequential catalysed reporter deposition (2C-FISH for the flow cytometric sorting of freshwater ultramicrobacteria

    Stefan M Neuenschwander


    Full Text Available Flow cytometric sorting is a powerful tool to physically separate cells within mixed microbial communities. If combined with phylogenetic staining (fluorescence in situ hybridization, FISH it allows to specifically sort defined genotypic microbial populations from complex natural samples. However, the targeted enrichment of freshwater ultramicrobacteria, such as members of the LD12 clade of Alphaproteobacteria (SAR11-IIIb, is still challenging. Current FISH protocols, even in combination with signal amplification by catalysed reporter deposition (CARD, are not sufficiently sensitive for the distinction of these bacteria from background noise by flow cytometry, presumably due to their low ribosome content and small cell sizes. We, therefore, modified a CARD based flow sorting protocol with the aim of increasing its sensitivity to a level sufficient for ultramicrobacteria. This was achieved by a second signal amplification step mediated by horseradish peroxidase labelled antibodies targeted to the fluorophores that were previously deposited by CARD-FISH staining. The protocol was tested on samples from an oligo-mesotrophic lake. Ultramicrobacteria affiliated with LD12 Alphaproteobacteria could be successfully sorted to high purity by flow cytometry. The ratios of median fluorescence signal to background ranged around 20, and hybridization rates determined by flow cytometry were comparable to those obtained by fluorescence microscopy. Potential downstream applications of our modified cell staining approach range from the analysis of microdiversity within 16S rRNA-defined populations to that of functional properties, such as the taxon-specific incorporation rates of organic substrates.

  1. Detection of P-glycoprotein with a rapid flow cytometric functional assay using Fluo-3: evaluation of sensitivity, specificity and feasibility in multiparametric analysis.

    Van Acker, K L; De Greef, C; Eggermont, J; Zhang, P; Vandenberghe, P; Boogaerts, M A


    The specificity and sensitivity of a flow cytometric assay simultaneously measuring expression and transport function of the multidrug resistance associated P-glycoprotein (Pgp) was evaluated. The monoclonal antibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional transport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CCRF-CEM/VLB50) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different substances (verapamil, vinblastine, trifluoperazine, cyclosporin A, progesterone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive counterparts. Unexpectedly, modulator activity was also observed in drug sensitive K562 and CCRF-CEM cell lines despite the inability to detect Pgp in those cells by MRK16 flow cytometrically. Low level expression of the MDR1 gene encoding Pgp in sensitive K562 cells was however demonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed to low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on Pgp function was more pronounced in a subpopulation of Pgp expressing lymphocytes than in lymphocytes which did not express Pgp. Finally, a correlation was found between discrete variations in Pgp expression and Pgp function of CD4+ lymphocytes, underscoring the feasibility of the functional assay in a triple parametric procedure. The triple parametric assay holds promise to detect Pgp expression and function in clinical samples containing mixtures of malignant and non-malignant cells.

  2. Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

    Werner, Kristin; Kälble, Solveig; Wolter, Sabine; Schneider, Erich H; Buschauer, Armin; Neumann, Detlef; Seifert, Roland


    The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

  3. Detection of chromosome aneuploidy in breast lesions with fluorescence in situ hybridization: Comparison of whole nuclei to thin tissue sections and correlation with flow cytometric DNA analysis

    Visscher, D.W.; Wallis, T.; Ritchie, C.A. [Wayne State Univ., Detroit, MI (United States)


    We compared flow-cytometric DNA histogram pattern to counts of 4 fluorescent-labelled centromeric probes (chromosomes 1, 7, 8, and 17) in whole nuclei (WN) and in nuclei from formalin-fixed deparaffinized thin tissue section (TS) in 25 breast lesions. In benign lesions, signal gains (i.e., trisomic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (0-43%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chromosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal losses in benign TS, on average, were 50-150% greater than in matched WN preparations (chromosome 1: 21.7%, chromosome 7: 21.5%). Malignant lesions generally, but not always, displayed fewer monosomic nuclei and more trisomic nuclei in compared to TS, compatible with a slicing (i.e., nuclear truncation) artifact. Signal counts in carcinomas correlated well with flow cytometric DNA index; however, they were also characterized by evidence of genetic instability, manifest as signal gains in a subset of nuclei (10-25%) with individual probes in diploid range cases, as well as intratumoral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples. We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The minimum signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability. 10 refs., 1 fig., 3 tabs.

  4. Flow Cytometric DNA Analysis Using Cytokeratin Labeling for Identification of Tumor Cells in Carcinomas of the Breast and the Female Genital Tract

    Rainer Kimmig


    Full Text Available Flow cytometric assessment of DNA‐ploidy and S‐phase fraction in malignant tumors is compromised by the heterogeneity of cell subpopulations derived from the malignant and surrounding connective tissue, e.g., tumor, stromal and inflammatory cells. To evaluate the effect on quality of DNA cell cycle analysis and determination of DNA ploidy, cytokeratin labeling of epithelial cells was used for tumor cell enrichment in breast, ovarian, cervical and endometrial cancer prior to DNA analysis. In a prospective study, tumor cell subpopulations of 620 malignant tumors were labeled by a FITC‐conjugated cytokeratin antibody (CK 5, 6, CK18 and CK 5, 6, 8 and CK 17, respectively prior to flow cytometric cell cycle analysis. Compared to total cell analysis, detection rate of DNA‐aneuploid tumors following cytokeratin labeling was increased from 62% to 76.5% in breast cancer, from 68% to 77% in ovarian cancer, from 60% to 80% in cervical cancer and from 30% to 53% in endometrial cancer. Predominantly in DNA‐diploid tumors, a significantly improved detection of S‐phase fraction of the tumor cells was shown due to the elimination of contaminating nonproliferating “normal cells”. S‐phase fraction following tumor cell enrichment was increased by 10% (mean following cytokeratin staining in ovarian and endometrial cancer, by 30% in breast cancer and even by 70% in cervical cancer compared to total cell analysis. Thus, diagnostic accuracy of DNA‐analysis was enhanced by cytokeratin labeling of tumor cells for all tumor entities investigated.

  5. Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

    Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K


    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

  6. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes; Analise por citometria de fluxo da expressao da proteina p21 em linfocitos humanos irradiados

    Santos, N.F.G.; Amaral, A., E-mail: [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Energia Nuclear. Laboratorio de Modelagem e Biodosimetria Aplicada; Freitas-Silva, R. [Universidade Federal de Pernambuco (UFPE), Garanhuns, PE (Brazil). Departamento de Ciencias Naturais e Exatas; Pereira, V.R.A. [Fundacao Oswaldo Cruz (FIOCRUZ), Recife, PE (Brazil). Centro de Pesquisas Aggeu Magalhaes. Departamento de Imunologia. Lab. de Imunoparasitologia; Tasat, D.R. [Universidad Nacional de General San Martin, Buenos Aires (Argentina). Escuela de Ciencia y Tecnologia. Laboratorio de Biologia Celular del Pulmon


    Cell cycle blockage in G1 is a mechanism p21 protein-regulated and coupled to DNA damage response to permit genetic content analysis, damage repair and cell death. Analysis of proteins that participates of this response has progressed with new analytic tools, and data contributes to comprehension of radioinduced molecular events as well as to new approaches on practices that employ ionizing radiation. On this perspective, the aim of this research was to evaluate, by flow cytometry, p21 expression on irradiated human lymphocytes, maintained under different experimental conditions. Peripheral blood samples from 10 healthy subjects were irradiated with doses of 0 (non-irradiated), 1, 2 and 4 Gy. Lymphocytes were processed to analysis on ex vivo (no cultured) condition and after 24; 48 and 72 hours culture, with and without phytohemagglutinin stimulation. p21 protein expression levels were measured by flow cytometry, as percentage values. Results indicate that flow cytometric assay allows detection of changes on p21 expression, since it was detected significant increase on phytohemagglutinin-stimulated samples, for all times, against basal expression (ex vivo). However, it was not observed significant alterations on p21 protein radioinduced levels, for all doses, times and culture conditions analyzed. These results not indicate so p21 protein as bioindicator of ionizing radiation exposure. Nevertheless, data confirmation may to require analysis of a more numerous population. (author)

  7. Development of flow cytometric protocol for nuclear DNA content estimation and determination of chromosome number in Pongamia pinnata L., a valuable biodiesel plant.

    Ramesh, Aadi Moolam; Basak, Supriyo; Choudhury, Rimjhim Roy; Rangan, Latha


    The potentiality of Pongamia pinnata L. as a sustainable source of feedstock for the biodiesel industry is dependent on an extensive knowledge of the genome structure of the plant. Flow cytometry, with propidium iodide (PI) as the DNA stain, was used to estimate the nuclear DNA content of P. pinnata, with respect to Zea mays 'CE-777' as standard. The internal and pseudo-internal standardization was followed on account of the inhibitory effect of secondary compounds on PI intercalation. The antioxidants (PVP-40 and β-mercaptoethanol) were added to the nuclear isolation buffer for the reduction of inhibitory effect of P. pinnata cytosol. Nuclear DNA content estimation was done for P. pinnata leaves from different altitudes (37-117 m height from sea level) of Assam. Flow cytometry analysis indicated that the nuclear DNA content of P. pinnata is 2.66 pg with predicted 1C value of 1,300 Mb using Z. mays as standard. Coefficient of variation in flow cytometric analysis was within the limit of 5 % indicating that the results were reliable. Somatic chromosome numbers were counted from root-tip cells and was found to be 2n = 22 corresponding to the diploid level (x = 11). A decreasing trend in the nuclear DNA content was observed for the species of different altitudes.

  8. Establishment of flow cytometric in micronucleus assay in vitro%流式细胞术检测体外微核方法的建立

    欧红梅; 周长慧; 涂宏刚; 黄鹏程; 常艳


    OBJECTIVE:Establish the flow cytometric 96-well microplate-basedin vitro micronucleus assay in CHO-K1 cells,and explore the possibility of this method for early genetic toxicity screening during drug discovery. MEHTODS:The test included treatment with and without metabolic activation. For the treatment with metabolic activation,CHO-K1 cells were treated with three different concentrations of cyclophosphamide in the S9 mixmedium for 4 h,then incubated with S9-free fresh medium for 20 h. For the treatment without metabolic activation,cells were incubated with three different concentrations of mitomycin C continuously for 24 h. In all cases,after a total of 24 h since initiation of the treatment,cells were processed for microscopic scoring or flow cytometric MN analysis. A flow cytometric method for scoring MN used EMA and SYTOX Green to label the cells in 96-well microplate,and then compared with cytokinesis-block micronucleus assay in cell culture disks based on microscopy.RESULTS:Mitomycin C and cyclophosphamide at different concerntrations caused statistically significant and dose-dependent increasess in micronucleus assay . Non-parametric Spearman's coefficients (rs) is 1.000.CONCLUSION:Similar to literature published,mitomycin C and cyclophosphamide induced positive results in flow cytometric based in vitro micronucleus assay. So the method of flow cytometric 96-well microplate-based in vitro micronucleus assay in CHO-K1 cells was established. The concordance between microscopic scoring and flow cytometricwas good,therefore this method is promising for screening and evaluating genetic toxicity of chemicals.%目的:建立96孔板流式细胞术体外微核自动化检测的方法,并探讨其用于药物早期遗传毒性筛选和遗传毒性评价的可能性。方法:试验分为+S9短时处理组(4 h)和-S9持续处理组(24 h),分别选择3个不同浓度的环磷酰胺和丝裂霉素C处理CHO-K1细胞,24 h后收获细胞。采用EMA和SYTOX Green

  9. Flow cytometric assay to assess short-term effects of personal care products on the marine microalga Tetraselmis suecica.

    Seoane, Marta; Esperanza, Marta; Rioboo, Carmen; Herrero, Concepción; Cid, Ángeles


    Large quantities of personal care products (PCPs) are used daily and many of their chemical ingredients are subsequently released into marine environments. Cultures of the marine microalga Tetraselmis suecica were exposed for 24 h to three emerging compounds included in the main classes of PCPs: the UV filter benzophenone-3 (BP-3), the disinfectant triclosan (TCS) and the fragrance tonalide (AHTN). Concentrations tested, expressed as cellular quota (pg cell(-1)), ranged from 5 to 40 for BP-3, from 2 to 16 for TCS and from 1.2 to 2.4 for AHTN. A small cytometric panel was carried out to evaluate key cytotoxicity biomarkers including inherent cell properties, growth and metabolic activity and cytoplasmic membrane properties. BP-3 caused a significant increase in growth rate, metabolic activity and chlorophyll a fluorescence from 10 pg cell(-1). However, growth and esterase activity decreased in cells exposed to all TCS and AHTN concentrations, except the lowest ones. Also these two compounds provoked a significant swelling of cells, more pronounced in the case of TCS-exposed cells. Although all treated cells remained viable, changes in membrane potential were observed. BP-3 and AHTN caused a significant depolarization of cells from 10 to 1.6 pg cell(-1), respectively; however all TCS concentrations assayed caused a noticeable hyperpolarization of cells. Metabolic activity and cytoplasmic membrane potential were the most sensitive parameters. It can be concluded that the toxicological model used and the toxicological parameters evaluated are suitable to assess the toxicity of these emerging contaminants.

  10. Flow cytometric analysis of kappa and lambda light chain expression in endoscopic biopsy specimens before the diagnosis of B-cell lymphoma.

    Oka, Satoko; Muroi, Kazuo; Sato, Kazuya; Fujiwara, Shin-ichiro; Oh, Iekuni; Matsuyama, Tomohiro; Ohmine, Ken; Suzuki, Takahiro; Ozaki, Katsutoshi; Mori, Masaki; Nagai, Tadashi; Fukushima, Noriyoshi; Fukushima, Noriyoshi; Tanaka, Akira; Ozawa, Keiya


    Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained.

  11. Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

    Dekking, E H A; van der Velden, V H J; Varro, R; Wai, H; Böttcher, S; Kneba, M; Sonneveld, E; Koning, A; Boeckx, N; Van Poecke, N; Lucio, P; Mendonça, A; Sedek, L; Szczepański, T; Kalina, T; Kanderová, V; Hoogeveen, P; Flores-Montero, J; Chillón, M C; Orfao, A; Almeida, J; Evans, P; Cullen, M; Noordijk, A L; Vermeulen, P M; de Man, M T; Dixon, E P; Comans-Bitter, W M; van Dongen, J J M


    The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.

  12. A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux.

    Bailey, Sheree; Macardle, Peter J


    Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca(2+) flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca(2+) flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca(2+) flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium-Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca(2+) flux was examined in response to membrane receptor stimulation or intracellular Ca(2+) mobilization. Dose dependent Ca(2+) flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca(2+) flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.

  13. Identification of New Rat Bone Marrow-Derived Population of Very Small Stem Cell with Oct-4A and Nanog Expression by Flow Cytometric Platforms

    Anna Labedz-Maslowska


    Full Text Available Very small embryonic-like stem cells (VSELs represent a unique rare population of adult stem cells (SCs sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM- derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS, we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

  14. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants



    Full Text Available Abstract. Teixeira da Silva JA. 2015. Response of Syngonium podophyllum L. ‘White Butterfly’ shoot cultures to alternative media additives and gelling agents, and flow cytometric analysis of regenerants. Nusantara Bioscience 7: 26-32. Syngonium podophyllum L. (arrowhead vine is a popular leafy indoor pot plant whose tissue culture has been established, primarily through in vitro shoot culture, but several interesting aspects have not yet been explored. In this study, cv. ‘White Butterfly’ was used to investigate the response of shoot formation to alternative gelling agents and media additives. Gellan gum (Gelrite® at 2 g/L resulted in greater leaf production, plantlet fresh weight and higher chlorophyll content (SPAD value than all other gelling agents tested, including agar, Bacto agar, phytagel, oatmeal agar, potato dextrose agar, barley starch and corn starch, when on a basal Hyponex® (NPK = 6.5: 6: 19; 3 g/L medium. Several alternative liquid medium additives tested (low and full fat milk, Coca-Cola®, coffee, Japanese green, Oolong and Darjeeling teas negatively impacted plant growth, stunted roots and decreased chlorophyll content (SPAD value of leaves. Plant growth on medium with refined sucrose or table sugar responded similarly. Poor growth was observed when crude extract from a high rebaudioside-containing stevia (Stevia rebaudiana Bertoni line - an artificial sweetener - was used. Leaf tissue from the control did not show any endopolyploidy but low levels of endopolyploidy (8C were detected in some treatments.

  15. The level of heparin-induced antibodies in correlation with the result of the flow cytometric functional assay in the patients with suspected HIT.

    Maličev, Elvira; Maček Kvanka, Marjeta; Klemenc, Polona; Rožman, Primož


    Heparin can induce the formation of antibodies against a heparin complex with a platelet factor 4 (PF4), leading to platelet activation and the development of heparin-induced thrombocytopaenia (HIT). Because screening ELISA does not discriminate between platelet activating and non-activating anti-heparin/PF4 antibodies, each positive result is confirmed by an additional functional assay. We analysed 1004 sera of patients with suspected HIT. Optical density (OD) values of ELISA-positive results were correlated with the risk for a positive result with our functional flow cytometric assay. Only 10.7% were ELISA positive and 59.8% of those were positive with the functional assay. The positive functional assay was found in 23.4% of patients with OD2.0. Although our results showed that higher ELISA OD values increasethe possibility of the presence of platelet-activating anti-heparin/PF4 antibodies - , there is no need for improving ELISA cut-off value for positive result. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  16. Flow cytometric assessment of circulating platelet and erythrocytes microparticles in young thalassemia major patients: relation to pulmonary hypertension and aortic wall stiffness.

    Tantawy, Azza A G; Adly, Amira A M; Ismail, Eman A R; Habeeb, Nevin M


    Heart disease is the leading cause of mortality and morbidity in β-thalassemia major (β-TM). Aggregability of abnormal red cells and membrane-derived microparticles (MPs) stemming from activated platelets and erythrocytes are responsible for thrombotic risk. We measured platelet and erythrocyte MPs (PMPs and ErMPs) in 60 young β-TM patients compared with 40 age- and sex-matched healthy controls and assessed their relation to clinicopathological characteristics and aortic elastic properties. Patients were studied stressing on transfusion history, splenectomy, thrombotic events, chelation therapy, hematological and coagulation profiles, flow cytometric measurement of PMPs (CD41b(+) ) and ErMPs (glycophorin A(+) ) as well as echocardiographic assessment of aortic elastic properties. Aortic stiffness index and pulmonary artery pressure were significantly higher, whereas aortic strain and distensibility were lower in TM patients than controls (P 2500 μg/L (P < 0.001). Compliant patients on chelation therapy had lower MPs levels than non-compliant patients (P < 0.001). PMPs and ErMPs were positively correlated to markers of hemolysis, serum ferritin, D-dimer, vWF Ag, and aortic stiffness, whereas negatively correlated to hemoglobin level and aortic distensibility (P < 0.05). We suggest that increased MPs may be implicated in vascular dysfunction, pulmonary hypertension risk, and aortic wall stiffness observed in thalassemia patients. Their quantification could provide utility for early detection of cardiovascular abnormalities and monitoring the biological efficacy of chelation therapy.

  17. A Simple Flow-Cytometric Method Measuring B Cell Surface Immunoglobulin Avidity Enables Characterization of Affinity Maturation to Influenza A Virus

    Frank, Gregory M.; Ince, William L.; Gibbs, James S.; Khurana, Surender; Wheatley, Adam K.; Max, Edward E.; McDermott, Adrian B.; Golding, Hana; Stevens, James; Bennink, Jack R.


    ABSTRACT Antibody (Ab) affinity maturation enables an individual to maintain immunity to an increasing number of pathogens within the limits of a total Ig production threshold. A better understanding of this process is critical for designing vaccines that generate optimal Ab responses to pathogens. Our study describes a simple flow-cytometric method that enumerates virus-specific germinal center (GC) B cells as well as their AC50, a measure of Ab avidity, defined as the antigen concentration required to detect 50% of specific B cells. Using a model of mouse Ab responses to the influenza A virus hemagglutinin (IAV HA), we obtained data indicating that AC50 decreases with time postinfection in an affinity maturation-dependent process. As proof of principle of the utility of the method, our data clearly show that relative to intranasal IAV infection, intramuscular immunization against inactivated IAV in adjuvant results in a diminished GC HA B cell response, with increased AC50 correlating with an increased serum Ab off-rate. Enabling simultaneous interrogation of both GC HA B cell quantity and quality, this technique should facilitate study of affinity maturation and rational vaccine design. PMID:26242629

  18. Rapid detection of BCR-ABL fusion genes using a novel combined LUX primer, in-cell RT-PCR and flow cytometric method.

    Shi, Yan; Li, Li-Zhen; Sun, Jian-Zhi; Zhang, Ti; Peng, Jun; Xu, Cong-Gao


    Currently, quantitative and semiquantitative assays for minimal residual disease detection include fluorescence in situ hybridisation, multiparameter flow cytometric immunophenotyping and real-time quantitative polymerase chain reaction (RQ-PCR). We have developed a new approach to detect hybrid breakpoint cluster region and Abelson proto-oncogene (BCR-ABL) transcripts inside suspension cells using in situ RT-PCR and light upon extension (LUX) primer, followed by rapid quantitative analysis with flow cytometry. After cellular permeabilization and fixation of single cell suspension, the neoplastic mRNA was reverse transcribed and amplified by PCR with LUX primer. The results demonstrated that a strong positive yellow-green signal was observed in 99-100% cells of K562 cell line, only the red nucleus was detected in NB4 cell line and normal controls. The technique has been utilised to study 12 patients with chronic myeloid leukemia, and the results were compared with those of BCR-ABL fusion mRNA by RT-PCR and BCR-ABL fusion gene of the interphase cells by fluorescence in situ hybridization (FISH). In the five diagnosed patients, 90-98% cells were strongly positive. Four patients, including three patients treated with interferon-alpha and hydroxyurea and one patient treated with imatinib mesylate, had 26-82.5% positive cells. Three patients treated with imatinib mesylate were negative. The in situ RT-PCR results demonstrated complete concordance with the results of I-FISH and RT-PCR. A fluorescence signal was detectable at 1/10(4) cells and became negative below this threshold with flow cytometry. The results of the present study suggest that (1) LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR; (2) the novel technique is a specific and sensitive way of detecting fusion gene with potential clinical usefulness.

  19. Flow cytometric detection of neutrophil-associated immunoglobulin in patients with or without neutropenia and establishment of the reference interval.

    Hwang, Keumrock; Park, Chan-Jeoung; Huh, Hee Jin; Han, Sang Hee; Jang, Seongsoo; Chi, Hyun-Sook


    We measured neutrophil-associated immunoglobulin (NAIg) levels using flow cytometry to establish the reference interval for NAIg and to estimate NAIg in patients with or without neutropenia. Peripheral blood from 152 individuals was analyzed for NAIg detection by flow cytometry. Using fluorescescent-conjugated anti-CD10 monoclonal antibody and anti-human immunoglobulins, proportions of NAIgG, NAIgA, and NAIgM bound to neutrophils were measured. Reference intervals for NAIg were set as NAIgG reference intervals defined herein, patients with neutropenia or adverse transfusion reactions may be evaluated in a clinically relevant manner.

  20. Development of a flow cytometric method to analyze subpopulations of bacteria in probiotic products and dairy starters

    Bunthof, C.J.; Abee, T.


    Flow cytometry (FCM) is a rapid and sensitive technique that can determine cell numbers and measure various physiological characteristics of individual cells by using appropriate fluorescent probes. Previously, we developed an FCM assay with the viability probes carboxyfluorescein diacetate (cFDA) a

  1. Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

    Peters, J.; Ploum, M.E.; Rijk, de T.C.; Haasnoot, W.


    A multi-mycotoxin immunoassay—using the MultiAnalyte Profiling (xMAP) technology—is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol,

  2. Stereologic, histopathologic, flow cytometric, and clinical parameters in the prognostic evaluation of 74 patients with intraoral squamous cell carcinomas

    Bundgaard, T; Sørensen, Flemming Brandt; Gaihede, M


    BACKGROUND AND METHODS: A consecutive series of all 78 incident cases of intraoral squamous cell carcinoma occurring during a 2-year period in a population of 1.4 million inhabitants were evaluated by histologic score (the modified classification of Jacobsson et al.), flow cytometry, stereology, ...

  3. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

    Rawstron, A C; Kennedy, B; Evans, P A; Davies, F E; Richards, S J; Haynes, A P; Russell, N H; Hale, G; Morgan, G J; Jack, A S; Hillmen, P


    Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

  4. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    Bienenmann-Ploum, Monique E.; Huet, Anne-Catherine; Campbell, Katrina; Fodey, Terence L.; Vincent, Ursula; Haasnoot, Willem; Delahaut, Philippe; Elliott, Christopher T; Nielen, Michel W. F.


    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microsph...

  5. Flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of Nile tilapia

    Segovia, M.; Jenkins, J.A.; Paniagua-Chavez, C.; Tiersch, T.R.


    Improved techniques for storage and evaluation of fish sperm would enhance breeding programs around the world. The goal of this study was to test the effect of antibiotics on refrigerated sperm from Nile tilapia (Oreochromis niloticus) by use of flow cytometry with 2 dual-staining protocols for objective assessment of sperm quality. Concentrations of 1 x 109 sperm/mL were suspended in Ringer's buffer at 318 mOsmol/kg (pH 8.0). The fluorescent stains Sybr 14 (10 ??M), propidium iodide (2.4 mM), and rhodamine 123 (0.13 ??M) were used to assess cell viability and mitochondrial function. Three concentrations of ampicillin, gentamicin, and an antibiotic/antimycotic solution were added to fresh spermatozoa. Motility estimates and flow cytometry measurements were made daily during 7 d of refrigerated storage (4 ??C). The highest concentrations of gentamicin and antibiotic/antimycotic and all 3 concentrations of ampicillin significantly reduced sperm viability. The highest of each of the 3 antibiotic concentrations significantly reduced mitochondrial function. This study demonstrates that objective sperm quality assessments can be made using flow cytometry and that addition of antibiotics at appropriate concentrations can lengthen refrigerated storage time for tilapia spermatozoa. With minor modifications, these protocols can be adapted for use with sperm from other species and with other tissue types.

  6. Direct detection of red blood cell fragments: a new flow cytometric method to evaluate hemolysis in blood pumps.

    Linneweber, J; Chow, T W; Takano, T; Maeda, T; Nonaka, K; Schulte-Eistrup, S; Kawahito, S; Elert, O; Moake, J L; Nosé, Y


    Pump induced hemolysis is presently evaluated by measuring plasma free hemoglobin (fHb). However, this method has disadvantages because quantification of fHb depends on hematocrit (HCT) and hemoglobin (Hb) levels. The aim of this work was to devise a hemoglobin independent method, capable of quantifying cell trauma directly by measuring the number of red blood cell (RBC) fragments. Whole blood flow cytometry was used to quantify circulating RBC fragments derived from a roller pump (Sarns, Inc. Model 2 M 6,002) and a centrifugal pump (Gyro C1E3, Kyocera Corp.). The pumps were tested in a mock circuit for 2 hr (5 L/min flow against 100 mm Hg pressure head). Red blood cell fragments were quantified by a phycoerythrin (PE) labeled glycophorin A antibody specific for erythrocytes. Red blood cell fragments were smaller than the intact RBC population and overlapped in size with the platelet population (based on forward- and side-light scattering measurements). For the roller pump, the values for RBC fragments increased from 1,090 +/- 260/microl at 0 min to 14,880 +/- 5,900/microl after 120 min. In contrast, using the centrifugal pump, there was little increase in RBC fragments (from 730 +/- 270/microl at 0 min to 1,400 +/- 840/microl after 120 min). Flow cytometry can be used for the rapid, sensitive, hemoglobin independent evaluation of pump induced RBC trauma.

  7. Improved flow cytometric identification of myelopoiesis by the simultaneous labelling with CD13, CD14 and CD66 monoclonal antibodies

    Bonde, J; Meyer, K; Broe, M K


    in the fast determination of remission state. In MDS, the immature myeloid component could be distinguished in patients defined according to the FAB classification with the possibility of identifying aberrant phenotypes, the assay should also be of interest in other myeloproliferative disorders. Moreover......The aim of the present study was to increase our knowledge of myelopoiesis evaluated by flow cytometry. We therefore designed a triple-marker assay employing monoclonal antibodies against the CD13 (immature), the CD14 (monocytic), and the CD66 (mature myeloid) antigens using three...

  8. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs.

    Bienenmann-Ploum, Monique E; Huet, Anne-Catherine; Campbell, Katrina; Fodey, Terence L; Vincent, Ursula; Haasnoot, Willem; Delahaut, Philippe; Elliott, Christopher T; Nielen, Michel W F


    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.

  9. Level 2 validation of a flow cytometric method for detection of Escherichia coli O157:H7 in raw spinach.

    Williams, Anna J; Cooper, Willie M; Summage-West, Christine V; Sims, Lillie M; Woodruff, Robert; Christman, Jessica; Moskal, Ted J; Ramsaroop, Shawn; Sutherland, John B; Alusta, Pierre; Wilkes, Jon G; Buzatu, Dan A


    The Bacteriological Analytical Manual (BAM) method currently used by the United States Food and Drug Administration (FDA) to detect Escherichia coli O157:H7 in spinach was systematically compared to a new flow cytometry based method. This Food and Drug Administration (FDA) level 2 external laboratory validation study was designed to determine the latter method's sensitivity and speed for analysis of this pathogen in raw spinach. Detection of target cell inoculations with a low cell count is critical, since enterohemorrhagic strains of E. coli require an infective dose of as few as 10 cells (Schmid-Hempel and Frank, 2007). Although, according to the FDA, the infectious dose is unknown (Food and Drug Administration, 1993). Therefore, the inoculation level into the spinach, a total of 2.0±2.6 viable E. coli O157 cells, was specified to yield between 25% and 75% detection by the new method, out of 20 samples (10 positives and 10 negatives). This criterion was met in that the new method detected 60% of the nominally positive samples; the corresponding sensitivity of the reference method was 50%. For both methods the most likely explanation for false negatives was that no viable cells were actually introduced into the sample. In this validation study, the flow cytometry method was equal to the BAM in sensitivity and far superior in speed. Published by Elsevier B.V.

  10. Flow cytometric comparison of platelets from a whole blood and finger-prick sample: impact of 24 hours storage.

    Swanepoel, Albe C; Stander, Andre; Pretorius, Etheresia


    In this study, we investigate the validity and laboratory utility of flow cytometry when analyzing platelet activation by studying CD41, CD42b, CD62P and CD63. We compare flow cytometry results from citrated whole-blood and finger-prick samples directly after collection and also after storing both a finger-prick and whole-blood sample for 24 hours. Citrated whole-blood and finger-prick samples were taken from three healthy individuals on two occasions, and a total of 60,000 cells were analyzed for each of the four phycoerythrin-labeled monoclonal antibodies. Half of each sample was analyzed immediately after sampling while the other half was kept in the fridge at 6 °C for 24 hours before analysis. No significant difference was found between the sampling methods or the period of time before analysis. Results therefore suggest that an appropriately prepared finger-prick sample can be used for platelet function analysis, and samples can be stored for 24 hours in the fridge at 6 °C before analysis.

  11. Flow Cytometric Determination of the Expression of gp 51 Protein of Bovine Leukaemia Virus in Experimentally Infected Sheep

    Szczotka Maria


    Full Text Available The study was performed on lambs experimentally infected with bovine leukaemia virus (BLV. The presence of BLV antibodies in sera of infected animals was detected by agar gel immunodiffusion test and ELISA. Proviral DNA was detected by PCR and nested PCR. Dual-colour flow cytometry analysis was performed with the use of specific monoclonal antibodies against lymphocyte CD markers and gp51 viral envelope protein, followed by incubation with fluorescent-labelled secondary antibodies conjugated with FITC or PE. Gp51 viral envelope protein was detected in tumours caused by BLV infection. The BLV infection resulted in depletion of CD4+ lymphocytes, increase in CD8+ lymphocytes, and decrease in CD4+ to CD8+ ratio in infected sheep. Proliferation of IgM+ CD19+ cells was also detected. These cells had an immature character without tendency to differentiate, and their vitality was prolonged. Flow cytometry enabled detection of gp51 expression in sheep blood lymphocytes at the early stages of the infection, before detection of serum antibodies using ELISA.

  12. Flow cytometric minimal residual disease monitoring in children with acute lymphoblastic leukemia treated by regimens with reduced intensity

    A. M. Popov


    Full Text Available 191 consecutive unselected children with acute lymphoblastic leukemia aged from 1 to 16 years were enrolled in the study. Bone marrow samples were obtained at the time of initial diagnostics as well as at days 15 (n = 188, 36 (n = 191, and 85 (n = 187 of remission induction. Minimal residual disease (MRD was assessed by 6–10-color flow cytometry. Flow cytometry data at day 15 allowed distinguishing three patients groups with significantly different outcome (p ˂ 0.0001: 35.64 % patients with MRD < 0.1 % represented 5-year event-free survival (EFS of 100 %; 48.40 % cases with 0.1 % ≤ MRD< 10 % had EFS 84.6 ± 4.2 %; 15.96 % patients with very high MRD (≥ 10 % belonged to group with poor outcome (EFS 56.7 ± 9.0 %. At the end of remission induction (day 36 36 children (18.85 % with MRD higher than 0.1 % had significantly worse outcome compared to remaining ones (EFS 49.4 ± 9.0 and 93.5 ± 2.1 % respectively; p ˂ 0.0001. From a clinical standpoint it is relevant to evaluate both low-risk and high-risk criteria. Multivariate analysis showed that day 15 MRD data is better for low-risk patients definition while end-induction MRD is the strongest unfavorable prognostic factor.

  13. Flow cytometric quantification of all phases of the cell cycle and apoptosis in a two-color fluorescence plot.

    Christine Vignon

    Full Text Available An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.

  14. Flow cytometric assessment of chicken T cell-mediated immune responses after Newcastle disease virus vaccination and challenge

    Dalgaard, T. S.; Norup, L. R.; Pedersen, A.R.


    . Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied....... Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls....... Immune chickens from both lines had a significantly higher frequency of circulating γδ T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after...

  15. Genetic stock assessment of spawning arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content.

    Lockwood, S F; Bickham, J W


    Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting "C" values for a particular species.

  16. Flow cytometric determination of stem/progenitor content in epithelial tissues: an example from nonsmall lung cancer and normal lung.

    Donnenberg, Vera S; Landreneau, Rodney J; Pfeifer, Melanie E; Donnenberg, Albert D


    Single cell analysis and cell sorting has enabled the study of development, growth, differentiation, repair and maintenance of "liquid" tissues and their cancers. The application of these methods to solid tissues is equally promising, but several unique technical challenges must be addressed. This report illustrates the application of multidimensional flow cytometry to the identification of candidate stem/progenitor populations in non-small cell lung cancer and paired normal lung tissue. Seventeen paired tumor/normal lung samples were collected at the time of surgical excision and processed immediately. Tissues were mechanically and enzymatically dissociated into single cell suspension and stained with a panel of antibodies used for negative gating (CD45, CD14, CD33, glycophorin A), identification of epithelial cells (intracellular cytokeratin), and detection of stem/progenitor markers (CD44, CD90, CD117, CD133). DAPI was added to measure DNA content. Formalin fixed paraffin embedded tissue samples were stained with key markers (cytokeratin, CD117, DAPI) for immunofluorescent tissue localization of populations detected by flow cytometry. Disaggregated tumor and lung preparations contained a high proportion of events that would interfere with analysis, were they not eliminated by logical gating. We demonstrate how inclusion of doublets, events with hypodiploid DNA, and cytokeratin+ events also staining for hematopoietic markers reduces the ability to quantify epithelial cells and their precursors. Using the lung cancer/normal lung data set, we present an approach to multidimensional data analysis that consists of artifact removal, identification of classes of cells to be studied further (classifiers) and the measurement of outcome variables on these cell classes. The results of bivariate analysis show a striking similarity between the expression of stem/progenitor markers on lung tumor and adjacent tumor-free lung.

  17. Immune toxicity of TiO₂ under hypoxia in the green-lipped mussel Perna viridis based on flow cytometric analysis of hemocyte parameters.

    Wang, Youji; Hu, Menghong; Li, Qiongzhen; Li, Jiale; Lin, Daohui; Lu, Weiqun


    The combined effects of DO and TiO2 (mixed rutile/anatase phase, 7/3) on immune responses in Perna viridis were examined. Mussels were exposed to six combinations of oxygen levels (hypoxia: 1.5 mg O2l(-1), normoxia: 6.0 mg O2 l(-1)) and TiO2 concentrations (0, 2.5 mg l(-1) and 10 mg l(-1)) for 216 h. Mussels were sampled after 24h, 48h, 120 h and 216 h, and immune parameters of hemocytes, including mortality, phagocytosis, non-specific esterase, ROS production, lysosomal content and total hemocyte count were investigated using flow cytometric assay. Hemocyte mortality was higher under hypoxia than normoxia, and increased with TiO2 concentrations, but no interaction was found between DO and TiO2. Phagocytosis was reduced under hypoxia and decreased with TiO2 exposure, and the interactive effect between time and TiO2 was observed. The percentage of hemocytes showing non-specific esterase activity was lower under hypoxia, and decreased as TiO2 concentration increased with the significant interactive effect of DO and TiO2. ROS production and lysosomal content were lower under hypoxia and reduced as concentration of TiO2 increased, and interactive effect of DO and TiO2 on ROS was evident. THC was significantly affected by the interactive effect between TiO2 and DO, with higher values under normoxia in the presence of TiO2. The present study demonstrated that immune functions of P. viridis were influenced by both nano-TiO2 and hypoxia with some synergistic effects between the two stressors. This implies that DO has to be considered in the evaluation of the toxicity of nano-materials to bivalves. © 2013.

  18. Equal overall rejection rate in pre-transplant flow-cytometric cross-match negative and positive adult recipients in liver transplantation.

    Matinlauri, Irma H; Höckerstedt, Krister A; Isoniemi, Helena M


    T cell IgG flow-cytometric cross-matches (FCXM) using 48 stored pre-transplant patient serum samples and 40 stored serum samples collected 3 wk after liver transplantation and frozen spleen cells of cadaveric donors in 48 consecutive liver transplantations were performed retrospectively. T cell IgG FCXM using pre-transplant serum samples was compared with 46 complement-dependent lymphocytotoxic cross-matches (CDCXM) performed at the time of transplantation. Clinical relevance of these tests was evaluated in relation to acute rejection, 1-, 3- and 5-yr graft and patient survival. The incidence of positive FCXM was 33% (16 of 48) and 13% (six of 46) by CDCXM. The median time of acute rejection was 29 d after transplantation in FCXM positive group (range 13-101 d) and 22 d in FCXM negative group (range 7-157 d, NS). Rejection rate was similar in 16 pre-transplant FCXM positive patients (eight of 16, 50%) compared with six pre-transplant CDCXM positive patients (three of six, 50%; NS). Recipients having graft rejection tended to be more often pre-transplant FCXM positive (eight of 21, 38%) than CDCXM positive (three of 21, 14%), but the difference was not significant (p > 0.1). No difference was found in the positive predictive value in relation to acute rejection between positive FCXM and CDCXM (69% vs. 50%; NS). Furthermore there was no correlation between post-transplant positive FCXM and acute rejection. No difference was found between pre-transplant T cell IgG FCXM positive and negative recipients in relation to graft or patient survival. Our findings are supportive for little risk associated with preformed donor-specific antibodies in liver transplantation.

  19. Multiparameter flow cytometric analysis of CD4 and CD8 T cell subsets in young and old people

    Özcelik Dennis


    Full Text Available Abstract Background T cell-mediated immunity in elderly people is compromised in ways reflected in the composition of the peripheral T cell pool. The advent of polychromatic flow cytometry has made analysis of cell subsets feasible in unprecedented detail. Results Here we document shifts in subset distribution within naïve (N, central memory (CM and effector memory (EM cells defined by CD45RA and CCR7 expression in the elderly, additionally using the costimulatory receptors CD27 and CD28, as well as the coinhibitory receptors CD57 and KLRG-1, to further dissect these. Although differences between young and old were more marked in CD8 than in CD4 cells, a similar overall pattern prevailed in both. Thus, the use of all these markers together, and inclusion of assays of proliferation and cytokine secretion, may enable the construction of a differentiation scheme applicable to CD4 as well as CD8 cells, with the model (based on Romero et al. suggesting the progression N→CM→EM1→EM2→pE1→pE2→EM4→EM3→E end-stage non-proliferative effector cells. Conclusion Overall, the results suggest that both differences in subset distribution and differences between subsets are responsible for age-related changes in CD8 cells but that differences within rather than between subsets are more prominent for CD4 cells.

  20. Protease substrate profiling using bacterial display of self-blocking affinity proteins and flow-cytometric sorting.

    Sandersjöö, Lisa; Jonsson, Andreas; Löfblom, John


    Proteases are involved in fundamental biological processes and are important tools in both biotechnological and biomedical research. An important property of proteases is to discriminate among potential substrates. Here, a new method for substrate profiling of proteases is presented. The substrates are displayed between two anti-idiotypic affinity domains on the Gram-positive bacterium Staphylococcus carnosus. The first domain functions as a reporter tag and has affinity for a labeled reporter protein, whereas the second domain blocks the reporter tag from interacting with the reporter protein. Site-specific proteolysis of the substrate results in release of the blocking domain, enabling the reporter tag to bind the labeled reporter protein. Proteolysis is therefore reflected in reporter binding, which is quantified by flow cytometry. First, the method with tobacco etch virus protease (TEVp) is evaluated and then the substrate preference of matrix metalloprotease-1 (MMP-1) is determined using two libraries of around three million substrates each. Identified substrate peptides contained the previously reported motif (PXXXHy ) and on-cell determination of apparent kcat /KM revealed that the enriched substrate peptides are hydrolyzed six to eight-fold more efficiently than a previously reported substrate peptide. The method thus works as intended and the authors believe it has potential as an efficient tool for substrate profiling.

  1. Picoplankton Community Composition by CARD-FISH and Flow Cytometric Techniques: A Preliminary Study in Central Adriatic Sea Water

    Anita Manti


    Full Text Available Data concerning picoplanktonic community composition and abundance in the Central Adriatic Sea are presented in an effort to improve the knowledge of bacterioplankton and autotrophic picoplankton and their seasonal changes. Flow cytometry analyses revealed the presence of two distinct bacteria populations: HNA and LNA cells. HNA cells showed an explicit correlation with viable and actively respiring cells. The study of viability and activity may increase our knowledge of the part that contributes really to the remineralization and bacterial biomass production. Authotrophic picoplankton abundance, especially picocyanobacteria, was strongly influenced by seasonality, indicating that light availability and water temperature are very important regulating factors. In terms of total carbon biomass, the main contribution came from heterotrophic bacteria with a lower contribution from autotrophic picoplankton. CARD-FISH evidenced, within the Eubacteria domain, the dominance of members of the phyla Alphaproteobacteria, with a strong contribution from SAR11clade, followed by Cytophaga-Flavobacterium and Gammaproteobacteria. The bacterial groups detected contributed differently depending when the sample was taken, suggesting possible seasonal patterns. This study documents for the first time picoplankton community composition in the Central Adriatic Sea using two different approaches, FCM and CARD-FISH, and could provide preliminary data for future studies.

  2. Transmission Electron Microscopic Morphological Study and Flow Cytometric Viability Assessment of Acinetobacter baumannii Susceptible to Musca domestica cecropin

    Shuiqing Gui


    Full Text Available Multidrug-resistant (MDR Acinetobacter baumannii infections are difficult to treat owing to the extremely limited armamentarium. Expectations about antimicrobial peptides' use as new powerful antibacterial agents have been raised on the basis of their unique mechanism of action. Musca domestica cecropin (Mdc, a novel antimicrobial peptide from the larvae of Housefly (Musca domestica, has potently active against Gram-positive and Gram-negative bacteria standard strain. Here we evaluated the antibacterial activity of Mdc against clinical isolates of MDR-A. baumannii and elucidate the related antibacterial mechanisms. The minimal inhibitory concentration (MIC of Mdc was 4 μg/mL. Bactericidal kinetics of Mdc revealed rapid killing of A. baumannii (30 min. Flow cytometry using viability stain demonstrated that Mdc causes A. baumannii membrane permeabilization in a concentration- and time-dependent process, which correlates with the bactericidal action. Moreover, transmission electron microscopic (TEM examination showed that Mdc is capable of disrupting the membrane of bacterial cells, resulting in efflux of essential cytoplasmic components. Overall, Mdc could be a promising antibacterial agent for MDR-A. baumannii infections.

  3. Bivariate flow cytometric analysis of DNA content versus immunopositivity for ribonucleotide reductase M1 subunit in the cell cycle.

    Mangiarotti, R; Bottone, M G; Danova, M; Pellicciari, C


    Ribonucleotide reductase (RR) is a cytoplasmatic enzyme catalyzing the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. Its activity strongly correlates to the rate of DNA synthesis. By using a specific monoclonal antibody against the large M1 subunit of RR, we assessed the expression of M1-RR versus DNA content by dual-parameter flow cytometry. The aim of this paper was to compare the variations in the immunopositivity for M1-RR during the cell cycle to the positivity for other cell cycle markers identifying either proliferating cells (Ki-67 and PCNA) or quiescent cells (statin). To do this, normal human embryonic fibroblasts in different growth conditions as well as several other mammalian cell lines (rat C6 glioma cells; mouse 3T3 fibroblasts and B16 melanoma cells; human epithelial EUE cells and mammary carcinoma MCF-7 cells) were used. The expression of M1-RR antigen was found to correlate positively with the expression of Ki-67 and PCNA, and negatively with the expression of statin. During early G1 phase, M1-RR becomes detectable by specific antibodies relatively later compared to PCNA and Ki-67; therefore, the lack of immunopositivity for M1-RR cannot be taken as an absolute indication of cell quiescence in G0.

  4. Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

    Frankfurt, O.S. (Roswell Park Memorial Institute, Buffalo, NY (USA))


    A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to the drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.


    Gröbner, S; Franz, M; Hoberg, U; Wetzka, B; Schweizer, T


    The use of assisted reproductive technologies (ARTs) is increasing worldwide. In order to predict the rate of pregnancy after ART the DNA fragmentation index (DFI) of ejaculated spermatocytes may be a better marker than conventional semen quality parameters. Spermatocytes with fragmented DNA are associated with apoptotic stages and are characterized by a low DNA content. The subhaploid nuclei of DNA-damaged spermatocytes can be easily detected by flow cytometry. We here analyzed the percentage of subhaploid nuclei of semen samples from 163 patients aged 26 to 74 years who consulted one of the ten centres for reproductive medicine which routinely send sperm samples to our laboratory in order to determine special sperm parameters. The percentage of subhaploid nuclei indicating the DFI of spermatocytes did not correlate with age and sperm volume, but inversely correlated with sperm concentration and the percentage of motile spermatocytes. This is in concordance with previous studies which demonstrated that DNA damage of spermatozoa correlates with conventional semen quality parameters. Since DNA-damaged spermatocytes are associated with an impaired outcome of assisted conception technologies, this method could help to monitor sperm quality of subfertile men after measures to increase sperm quality and to improve selection criteria of cryopreserved sperm samples in assisted reproduction medicine.

  6. Immunophenotypic Characterization of Human Bone Marrow Mast Cells. A Flow Cytometric Study of Normal and Pathological Bone Marrow Samples

    Luis Escribano


    Full Text Available The goal of the present paper was to define the immunophenotype of bone marrow mast cells (BMMC from healthy controls and patients with hematologic malignancies (HM based on the use of multiple stainings with monoclonal antibodies analyzed by flow cytometry. Our results show that BMMC from both groups of individuals display a similar but heterogenous immunophenotype. The overall numbers of BMMC are higher in the HM group of individuals (p = 0.08. Three patterns of antigen expression were detected: (1 markers constantly positive in all cases analyzed (CD9, CD29, CD33, CD43, CD44, CD49d, CD49e, CD51, CD71, CD117, and FcεRI, (2 antigens that were constantly negative (CD1a, CD2, CD3, CD5, CD6, CD11a, CD14, CD15, CD16, CD19, CD20, CD21, CD23, CD25, CD30, CD34, CD38, CD41a, CD42b, CD65, CD66b, HLA-DR, and CD138, and (3 markers that were positive in a variable proportion of cases – CD11b (50%, CD11c (77%, CD13 (40%, CD18 (20%, CD22 (68%, CD35 (27%, CD40 (67%, CD54 (88% and CD61 (40%. In addition, BMMC from all cases explored were CD45+, and this antigen was expressed at an intensity similar to that of mature granulocytes.

  7. Flow cytometric assessment of reactive oxygen species generations that are directly related to cellular ZnO nanoparticle uptake.

    Yoo, Hyun Ju; Yoon, Tae Hyun


    In this study, a simple flow cytometry protocol to evaluate nanoparticle associated biological response was proposed. Particularly, we have evaluated the effect of surface charge on the cellular nanoparticle associations and nanoparticle-induced apoptosis. Significant enhancement in side scattering intensity was observed for the HeLa cells treated with positively charged (PLL)ZnO nanoparticles, suggesting that the (PLL)ZnO nanoparticles may induce cell death via adsorption and endocytosis of the nanoparticles. On the other hand, the negatively charged (PAA)ZnO nanoparticle seems to cause cell death process indirectly via the released Zn ions, with less contribution from cellular association of nanoparticles. Time- and dose-dependent studies on cellular association of ZnO nanoparticles, and ZnO associated reactive oxygen species generation were also performed for the HeLa cells exposed to the (PLL)ZnO nanoparticle. For those cells associated with (PLL)ZnO nanoparticle, a significant enhancement in reactive oxygen species generation was observed even at a lower concentration (10 ppm), which was not observable for the results with the whole cell population. By using this approach, we are able to distinguish biological responses (e.g., reactive oxygen species (ROS) generation) directly related to the cellular associations of NPs from those indirectly related to the cellular associations of NPs, such as the cytotoxicity caused by the NP released metal ions.

  8. Ultrastructural and flow cytometric analyses of lipid accumulation in microalgae: Annual report, Solar Energy Research Institute, Aquatic Species Program

    Solomon, J.A.; Hand, R.E. Jr.; Mann, R.C.


    Lipid accumulation in three species of microalgae was investigated with flow cytometry (FCM) and transmission electron microscopy (TEM). Previous studies using batch cultures of algae have led to the assumption that lipid accumulation in microalgae is a gradual process requiring at least several days for completion. However, FCM reveals, through changes in the chlorophyll:lipid ratio, that the time span required for individual cells to change metabolic state is short. Simultaneous FCM measurements of chlorophyll and nile red (neutral lipid) fluorescence in individual cells of nitrogen-deficient Isochrysis populations revealed a bimodal population distribution as one stage in the lipid accumulation process. The fact that two discrete populations exist, with few cells in an intermediate stage, suggests rapid response to a lipid trigger. Interpretations of light and electron microscopic observations are consistent with this hypothesis. The time required for an entire population to achieve maximum lipid content is considerably longer than that required for a single cell, due to the variation in response time among cells. In this study high lipid cultures were sometimes obtained by using FCM to separate high lipid cells from the remainder of the population. FCM holds much promise for strain enhancement but considerable developmental work, directed at providing more consistent results, remains to be done. 8 refs., 33 figs.

  9. Flow cytometric analysis of the H2O2-induced increase in intracellular Ca2+ concentration of rat thymocytes.

    Okazaki, E; Chikahisa, L; Kanemaru, K; Oyama, Y


    The effect of hydrogen peroxide (H2O2) on the intracellular Ca2+ concentration ([Ca2+]i) of rat thymocytes was examined by a flow cytometer and two fluorescent dyes, fluo-3-AM and ethidium bromide, a dye impermeant to intact membranes, to characterize the H2O2-induced increase in [Ca2+]i. H2O2 at concentrations greater than 30 microM dose-dependently increased the [Ca2+]i of thymocytes which were not stained with ethidium. Removal of external Ca2+ greatly reduced the degree of H2O2-induced increase in [Ca2+]i. However, H2O2 still increased the [Ca2+]i under the external Ca(2+)-free condition. Diethylmaleate, which is known to produce a chemical depletion of cellular nonprotein thiol, significantly increased the [Ca2+]i. Dithiothreitol, which is used to protect cellular nonprotein thiol, slightly decreased the [Ca2+]i, but greatly reduced the H2O2-induced increase in [Ca2+]i. Therefore, it is considered that H2O2 may increase the [Ca2+]i through a mechanism related to the effects of H2O2 on the cellular nonprotein thiol.

  10. Quality control of flow cytometric immunophenotyping%流式细胞术表型分析的质量控制

    吴丽娟; 许东升


    FCM表型分析已经从外周血淋巴细胞的双参数定量测定发展到当今对血液细胞病理学包括骨髓、淋巴结分析的5个或更多参数的定性测定,其中白血病和淋巴瘤的免疫表型分析在血液淋巴系统恶性疾病诊断、分类及病情监测上都是对形态学检验的一种重要补充.FCM 5个或更多参数分析的复杂性以及数据的解释依赖于仪器、试剂、程序的标准化及校准.此外,在美国的流式细胞学实验室还必须保留有关水平测试、样品制备、方法学的准确性、特异性、灵敏度和精密度的相关资料.CLSI和UCCC建议每个流式细胞学实验室需要对定性和定量检测程序进行验证.本文对美国临床流式细胞学实验室FCM检验的相关验证、室内与室间质量控制措施进行介绍.%Flow cytometric immunophenotyping has evolved from two-parameter quantitative measurement of peripheral blood lymphocytes to five-or more parameter qualitative evaluation of bone marrow and lymph node in hematopathology.Leukemia/lymphoma immunophenotyping represents an important addition to histomorphology in the diagnosis,classification and monitoring of hematolymphoid neoplasms.The complexity of five-or more parameter analyses and the interpretation of the data rely on standardization and validation of the instrument,the reagent and the procedure.In addition,clinical flow cytometry laboratories in U.S.A are required to document proficiency testing,sample preparation as well as accuracy,specificity,sensitivity and precision of methodology.CLSI and UCCC recommend that each laboratory should validate its own qualitative and quantitative procedures.This paper introduces the procedures for validation and quality control in a clinical flow cytometry laboratory in the United States.

  11. Flow cytometric analysis of the inhibition of human basophil activation by histamine high dilutions – a replication study

    Chantal Wälchli


    perform in order to reduce the possibility of artifacts but was omitted in the former study. Conclusions: Laboratory independent replication of homeopathic basic research experiments is still a challenge. Assuming that the results formerly obtained with this model were not due to systematic errors, the quest identifying the crucial factors for successful reproducibility is open for future research. Keywords: Human basophils; histamine; high dilutions; flow cytometry Reference: [1] Sainte-Laudy J, Belon P. Improvement of flow cytometric analysis of basophil activation inhibition by high histamine dilutions. A novel basophil specific marker: CD 203c. Homeopathy. 2006;95:3-8.

  12. Evaluation of zinc oxide nanoparticles toxicity on marine algae chlorella vulgaris through flow cytometric, cytotoxicity and oxidative stress analysis.

    Suman, T Y; Radhika Rajasree, S R; Kirubagaran, R


    The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnO NPs) was investigated in Marine algae Chlorella vulgaris (C. vulgaris). High zinc dissociation from ZnONPs, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONPs toxicity. To examine the mechanism of toxicity, C. vulgaris were treated with 50mg/L, 100mg/L, 200mg/L and 300 mg/L ZnO NPs for 24h and 72h. The detailed cytotoxicity assay showed a substantial reduction in the viability dependent on dose and exposure. Further, flow cytometry revealed the significant reduction in C. vulgaris viable cells to higher ZnO NPs. Significant reductions in LDH level were noted for ZnO NPs at 300 mg/L concentration. The activity of antioxidant enzyme superoxide dismutase (SOD) significantly increased in the C. vulgaris exposed to 200mg/L and 300 mg/L ZnO NPs. The content of non-enzymatic antioxidant glutathione (GSH) significantly decreased in the groups with a ZnO NPs concentration of higher than 100mg/L. The level of lipid peroxidation (LPO) was found to increase as the ZnO NPs dose increased. The FT-IR analyses suggested surface chemical interaction between nanoparticles and algal cells. The substantial morphological changes and cell wall damage were confirmed through microscopic analyses (FESEM and CM).

  13. Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

    HUANG Jian; WEN Ruobing; BAO Zhenmin; SUI Zhenghong; SUN Ningbo; KANG Kyoungho


    Over the last severaldecades,harmful algal blooms (HABs) havebecome a serious environmental problem in many parts of the world.A rapid and accurate detection process for HAB algae has yet to be developed.Heterosigma akashiwo is one of the most important HABs species in China.The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H.akashiwo cells.Five HABs species (Alexandrium catenella,Thalassiosira sp.,Cryptomonas sp.,Alexandrium tamarense and Symbiodinium sp.,) were used in this study to evaluate the analysis process we developed.A polyclonal antibody with high titers against H.akashiwo was obtained by injecting H.akashiwo cells into rabbits.Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H.akashiwo cells from the culture.Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs,and only 5.3%-12.5% of the four other HAB microalgae species were captured,indicating that the constructed IMBs combined specifically with the H.akashiwo cells.At the same time,flow cytometry (FCM) sorting was exploited to screen H.akashiwo cells after labeling with FITC conjugated polyclonal antibodies.Using the FCM technique,91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes.These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species.These techniques,especially immuno-magnetic separation,have low associated cost,and are fast and simple processes compared with other techniques currently in use.

  14. Flow-cytometric analysis of T-lymphocyte subsets after different treatment methods in patients with pericoronitis.

    Orbak, Recep; Dayi, Ertunç


    The aim of this study was to determine whether there was any change in T-lymphocyte subsets in patients with periocoronitis after the application of different treatment methods. Twenty-six patients with acute pericoronitis were included in the study. In every phase of the treatment (pretreatment, postcurettage, and postextraction), the biopsy samples were taken from the gingival tissues at sites of pericoronitis. Then, CD4(+) and CD8(+) lymphocyte and CD4(+)/CD8(+) ratio values were determined using flow cytometry in the biopsy samples. At the same time, gingival index (Löe-Silness) and plaque index (Silness-Löe) scores were recorded to assess the periodontal status in patients. To determine the correlation between the clinical measurements and the laboratory results obtained before the treatment, after curettage, and after extraction, we conducted an analysis using a paired t-test. The normal values in peripheral blood of CD4(+) and CD8(+) lymphocytes are 25% to 29% and 19% to 48%, respectively. However, the CD4(+) and CD8(+) lymphocyte values in the patients with acute pericoronitis were found to be 22.12% +/- 6.15% and 7.69% +/- 4.12%, respectively. These values are lower than the normal values. The CD4(+) lymphocyte value increased to 31.06% +/- 7.09% postcurettage and to 32.24% +/- 3.11% postextraction. The CD8(+) lymphocyte value increased to 16.21% +/- 5.27% postcurettage and to 18.25% +/- 3.13% postextraction. The CD4/CD8 ratio increased postcurettage and postextraction. This increase was statistically significant (P pericoronitis pathobiology.

  15. Modeling of inter-sample variation in flow cytometric data with the joint clustering and matching procedure.

    Lee, Sharon X; McLachlan, Geoffrey J; Pyne, Saumyadipta


    We present an algorithm for modeling flow cytometry data in the presence of large inter-sample variation. Large-scale cytometry datasets often exhibit some within-class variation due to technical effects such as instrumental differences and variations in data acquisition, as well as subtle biological heterogeneity within the class of samples. Failure to account for such variations in the model may lead to inaccurate matching of populations across a batch of samples and poor performance in classification of unlabeled samples. In this paper, we describe the Joint Clustering and Matching (JCM) procedure for simultaneous segmentation and alignment of cell populations across multiple samples. Under the JCM framework, a multivariate mixture distribution is used to model the distribution of the expressions of a fixed set of markers for each cell in a sample such that the components in the mixture model may correspond to the various populations of cells, which have similar expressions of markers (that is, clusters), in the composition of the sample. For each class of samples, an overall class template is formed by the adoption of random-effects terms to model the inter-sample variation within a class. The construction of a parametric template for each class allows for direct quantification of the differences between the template and each sample, and also between each pair of samples, both within or between classes. The classification of a new unclassified sample is then undertaken by assigning the unclassified sample to the class that minimizes the distance between its fitted mixture density and each class density as provided by the class templates. For illustration, we use a symmetric form of the Kullback-Leibler divergence as a distance measure between two densities, but other distance measures can also be applied. We show and demonstrate on four real datasets how the JCM procedure can be used to carry out the tasks of automated clustering and alignment of cell

  16. Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets, erythrocytes, and leukocytes.

    Pichler, J; Printz, D; Scharner, D; Trbojevic, D; Siekmann, J; Fritsch, G


    Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.

  17. Applications of immuno-magnetic bead and immunofluorescent flow cytometric techniques for the quantitative detection of HAB microalgae

    Huang, Jian; Wen, Ruobing; Bao, Zhenmin; Sui, Zhenghong; Sun, Ningbo; Kang, Kyoungho


    Over the last several decades, harmful algal blooms (HABs) have become a serious environmental problem in many parts of the world. A rapid and accurate detection process for HAB algae has yet to be developed. Heterosigma akashiwo is one of the most important HABs species in China. The objective of this study was to develop an immunologic technique that can rapidly and sensitively count H. akashiwo cells. Five HABs species ( Alexandrium catenella, Thalassiosira sp., Cryptomonas sp., Alexandrium tamarense and Symbiodinium sp.), were used in this study to evaluate the analysis process we developed. A polyclonal antibody with high titers against H. akashiwo was obtained by injecting H. akashiwo cells into rabbits. Immuno-magnetic beads (IMB) were produced via conjugated polyclonal antibodies with magnetic beads and applied to isolate and count H. akashiwo cells from the culture. Results show that 66.7%-91.6% of the cells were captured from unialgal culture by IMBs, and only 5.3%-12.5% of the four other HAB microalgae species were captured, indicating that the constructed IMBs combined specifically with the H. akashiwo cells. At the same time, flow cytometry (FCM) sorting was exploited to screen H. akashiwo cells after labeling with FITC conjugated polyclonal antibodies. Using the FCM technique, 91.7% of the targeted cells were sorted out from mixed microalgae samples in just a few minutes. These results indicate that both antibody-involved IMB and antibody-based FCM techniques are highly effective at detecting and quantifying HAB species. These techniques, especially immuno-magnetic separation, have low associated cost, and are fast and simple processes compared with other techniques currently in use.

  18. Flow cytometric analysis on tri-n-butyltin-induced increase in annexin V binding to membranes of rat thymocytes.

    Nakata, M; Oyama, Y; Okada, Y; Yamazaki, Y; Chikahisa, L; Satoh, M


    Effects of tri-n-butyltin chloride (TBT) on rat thymocytes were examined by using a flow cytometer and three fluorescent dyes (annexin V-FITC, ethidium bromide and fluo-3-AM) to further characterize its cytotoxic action. TBT at concentrations of 100 nM or greater, time- and dose-dependently increased the population of annexin V-positive live cells in the cell suspension. Most of cells became to be annexin V-positive within 60 min after the start of application of 300 nM TBT. Some of annexin V-positive live cells were further stained with ethidium, indicating that some of the cells were killed, in continued presence of TBT at 300 nM or greater. When the cells were exposed to 300 nM TBT only for 15 min, the population of annexin V-positive live cells increased after removal of TBT from incubation medium. TBT-induced increase in the population of annexin V-positive live cells was partly attenuated under Ca(2+)-free condition, although that was not the case for the dead cells. TBT at 30 nM or greater increased [Ca(2+)]i in a dose-dependent manner. Triethyltin and trimethyltin even at 1 μM did not increase the [Ca(2+)]i and the population of annexin V-positive live cells. The population of annexin V-positive live cells increased as the [Ca(2+)]i was increased by ionomycin, a calcium ionophore. Results suggest an involvement of Ca(2+) in some of TBT-induced cytotoxicity.

  19. Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

    Sahu, Saura C; Njoroge, Joyce; Bryce, Steven M; Zheng, Jiwen; Ihrie, John


    Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

  20. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.


    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  1. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    Jogdand, Prajakta S; Singh, Susheel K; Christiansen, Michael;


    ABSTRACT: BACKGROUND: Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays...... asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination...

  2. Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

    Peters, J.; Thomas, D.; Boers, E.A.M.; Rijk, de T.C.; Berthiller, F.; Haasnoot, W.; Nielen, M.W.F.


    A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins

  3. In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

    Hein-Kristensen, L; Wiese, L; Kurtzhals, J A L


    Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, pr...

  4. Colour-encoded paramagnetic microbead-based direct inhibition triplex flow cytometric immunoassay for ochratoxin A, fumonisins and zearalenone in cereals and cereal-based feed

    Peters, J.; Thomas, D.; Boers, E.A.M.; Rijk, de T.C.; Berthiller, F.; Haasnoot, W.; Nielen, M.W.F.


    A combined (triplex) immunoassay for the simultaneous detection of three mycotoxins in grains was developed with superparamagnetic colour-encoded microbeads, in combination with two bead-dedicated flow cytometers. Monoclonal antibodies were coupled to the beads, and the amounts of bound mycotoxins w

  5. Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition

    Jogdand Prajakta S


    Full Text Available Abstract Background Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos was used for obtaining reliable live parasite counts through flow cytometry. Methods Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI in an antibody-dependent cellular inhibition (ADCI assay. Results Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP

  6. Flow cytometric applications of tumor biology: prospects and pitfalls. [Applications in study of spontaneous dog tumors and in drug and radiation effects on cultured V79 cells

    Raju, M.R.; Johnson, T.S.; Tokita, N.; Gillette, E.L.


    A brief review of cytometry instrumentation and its potential applications in tumor biology is presented using our recent data. Age-distribution measurements of cells from spontaneous dog tumors and cultured cells after exposure to x rays, alpha particles, or adriamycin are shown. The data show that DNA fluorescence measurements have application in the study of cell kinetics after either radiation or drug treatment. Extensive and careful experimentation is needed to utilize the sophisticated developments in flow cytometry instrumentation.

  7. Flow Cytometric Panel-Reactive Antibody Results and the Ability to Find Transfusion-Compatible Platelets after Antibody-Desensitization for Allogeneic Bone Marrow Transplant.

    Rosenbaum, Eric R; Pandey, Soumya; Harville, Terry O; Drobena, Gina A; Cottler-Fox, Michele


    Panel reactive antibody (PRA) reduction protocols are used to decrease anti-HLA antibodies with concomitant PRA monitoring as a measure of successful treatment prior to organ and haploidentical blood and marrow transplant (BMT). We hypothesized that the more sensitive flow cytometry (FC) based assays for PRA [FlowPRA(®) and Luminex(®) based Single Antigen Bead (SAB)] would also correlate with the ability to find compatible platelets for allosensitized recipients. A female patient with myelodysplastic syndrome and a high HLA class I PRA [>90% PRA and cPRA by complement-dependent cytotoxicity (CDC) assay and Flow PRA] required allogeneic BMT. Baseline HLA Class I and class II antigen typing was performed and a matched sibling donor was identified. Although baseline anti-HLA class I and class II antibodies measured by FC and CDC revealed no donor specific antibodies (DSA), the decision was made to attempt antibody desensitization to facilitate platelet transfusion during BMT. FC and CDC assays were performed to determine anti-HLA class I antibodies and cPRA/%PRA prior to starting desensitization and at the end of desensitization. Over the course of desensitization and BMT, a total of 194 apheresis platelet units underwent cross-match (XM) using Capture-P(®). We compared temporally-related PRA results with platelet XM results. High PRA by FC or CDC assays correlates with a high % of XM-positive (incompatible) platelet units. When the CDC PRA fell to 2% after desensitization, platelet XM incompatibility fell from 100% to 63% positive (incompatible). When the FC PRA fell to 5% the positive platelet XM fell to 5%. Antibody desensitization facilitated platelet transfusion. PRA determination by FC appeared better correlated than determination by CDC with the ability to find XM-compatible platelets. © 2016 by the Association of Clinical Scientists, Inc.

  8. Super-resolved calibration-free flow cytometric characterization of platelets and cell-derived microparticles in platelet-rich plasma.

    Konokhova, Anastasiya I; Chernova, Darya N; Moskalensky, Alexander E; Strokotov, Dmitry I; Yurkin, Maxim A; Chernyshev, Andrei V; Maltsev, Valeri P


    Importance of microparticles (MPs), also regarded as extracellular vesicles, in many physiological processes and clinical conditions motivates one to use the most informative and precise methods for their characterization. Methods based on individual particle analysis provide statistically reliable distributions of MP population over characteristics. Although flow cytometry is one of the most powerful technologies of this type, the standard forward-versus-side-scattering plots of MPs and platelets (PLTs) overlap considerably because of similarity of their morphological characteristics. Moreover, ordinary flow cytometry is not capable of measurement of size and refractive index (RI) of MPs. In this study, we 1) employed the potential of the scanning flow cytometer (SFC) for identification and characterization of MPs from light scattering; 2) suggested the reference method to characterize MP morphology (size and RI) with high precision; and 3) determined the lowest size of a MP that can be characterized from light scattering with the SFC. We equipped the SFC with 405 and 488 nm lasers to measure the light-scattering profiles and side scattering from MPs, respectively. The developed two-stage method allowed accurate separation of PLTs and MPs in platelet-rich plasma. We used two optical models for MPs, a sphere and a bisphere, in the solution of the inverse light-scattering problem. This solution provides unprecedented precision in determination of size and RI of individual spherical MPs-median uncertainties (standard deviations) were 6 nm and 0.003, respectively. The developed method provides instrument-independent quantitative information on MPs, which can be used in studies of various factors affecting MP population.

  9. Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains.

    Booyens, Jemma; Thantsha, Mapitsi Silvester


    It is generally reported that garlic (Allium sativum) harms pathogenic but not beneficial bacteria. Although numerous studies supporting the alleged garlic effects on pathogens are available, there are limited studies to prove this claim for beneficial bacteria. We have recently shown that garlic exhibits antibacterial activity against probiotic bifidobacteria. The aim of the current study was to elucidate the mechanism of action of garlic clove extract (GCE) on Bifidobacterium bifidum LMG 11041, B. longum LMG 13197 and B. lactis Bb12 using Fourier transform infrared (FT-IR) spectroscopy and flow cytometry. Cultures (1 × 108 CFU ml-1) were individually incubated for 6 h at 37°C in garlic clove extract containing allicin at a corresponding predetermined minimum bactericidal concentration for each strain. For FTIR, an aliquot of each culture was deposited on CaF2 slide and vacuum dried. The slides were immediately viewed using a Bruker Vertex 70 V FT-IR spectrometer equipped with a Hyperion microscope and data analyzed using OPUS software (version 6, Bruker). Spectra were smoothed with a Savitsky-Goly function algorithim, base-line corrected and normalized. Samples for flow cytometry were stained using the Live/Dead BacLight bacterial viability kit L7012. Data compensation and analysis was performed using a BD FACSAria and FlowJo (version 7.6.1). Fourier transform infrared spectroscopy showed changes in spectral features of lipids and fatty acids in cell membranes, proteins, polysaccharides and nucleic acids. Spectral data as per principle component analysis (PCA) revealed segregation of control and GCE-treated cells for all the tested bifidobacteria. Flow cytometry not only showed increase in numbers of membrane damaged and possibly lysed cells after GCE treatment, but also displayed diffuse light scatter patterns for GCE treated cells, which is evidence for changes to the size, granularity and molecular content of the cells. Garlic has multiple target sites in

  10. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Anna Grazia Recchia

    Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  11. Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

    Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato


    Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

  12. Epstein-Barr Virus, Human Papillomavirus, and Flow Cytometric Cell Cycle Kinetics in Nasopharyngeal Carcinoma and Inverted Papilloma among Egyptian Patients

    S. K. Kassim


    Full Text Available It is widely accepted that the Epstein-Barr virus is etiologically associated with the development of nasopharyngeal carcinoma. The human papillomavirus is also associated with inverted papilloma. We used the polymerase chain reaction technique to detect both viruses in both types of tumors. Flow cytometry was also used to study the DNA pattern and proliferative behavior of the tumors in relation to the viruses. EBV was detected in 13/20 (65% of NPC specimens, and in none of IP (n = 10 or control specimens (n = 10. This indicates the contribution of EBV as an etiologic factor in NPC. Five cases of NPC (25% were positive for HPV 16, two of them were EBV positive. Four HPV 16 positive cases were found among cases with inverted papilloma, but none among the control cases. Flow cytometry revealed that all NPC, IP, and control samples were diploid except one aneuploid NPC sample. Proliferative capacity (PC of primary tumors was predictive of tumor recurrence in NPC. Using 13.6% as a cut-off point for PC, we were able to discriminate between high risk and low risk groups with 100% sensitivity and 86% specificity. PC can be used as a baseline prognostic parameter in NPC, making it possible to modify courses of treatment in an attempt to inhibit tumor recurrence.

  13. Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia.

    Miyazaki, T; Matsuda, I; Oguri, M; Amaya, H; Kiyosaki, M; Hamada, A; Tamaki, S; Tashiro, E; Kudo, Y; Taniguchi, O; Nakamura, T; Tomoyasu, S


    We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

  14. Single-colour flow cytometric assay to determine NK cell-mediated cytotoxicity and viability against non-adherent human tumor cells.

    Thakur, Ajit; Zaman, Abeyat; Hummel, Jeff; Jones, Kim; Hortelano, Gonzalo


    A flow cytometry-based cytotoxicity (FCC) assay was developed using a single fluorophore, calcein-acetoxymethyl diacetylester (calcein-AM), to measure NK cell-mediated cytotoxicity. Non-adherent human K562 and U937 target cells were individually labelled with calcein-AM and co-incubated with effector NK cells to measure calcein loss, and therefore calculate target cell cytotoxicity. This FCC assay also provided a measure of sample viability. Notably, cell viability measured by traditional calcein/7-amino-actinomycin D (7-AAD) double labelling and Trypan Blue methods were comparable to the viability calculated using calcein-loss FCC. This FCC assay may also be used with various effector and target cell types and as a multi-parameter tool to measure viability and immunophenotype cells for tissue engineering purposes.

  15. Flow cytometric detection and quantification of CD56 (neural cell adhesion molecule, NCAM) expression in diffuse large B cell lymphomas and review of the literature.

    Stacchini, Alessandra; Barreca, Antonella; Demurtas, Anna; Aliberti, Sabrina; di Celle, Paola Francia; Novero, Domenico


    To report unusual CD56 (neural cell adhesion molecule, NCAM) expression on diffuse large B cell lymphoma (DLBCL). CD56 expression was first detected and quantified on tissues obtained from five cases of DLBCL by flow cytometry (FC), then confirmed by immunohistochemistry. The CD56 expression pattern was heterogeneous among the cases [the molecular equivalent of soluble fluorochrome (MESF) level ranged from 2214 to 133 466]. All were CD10 and Bcl-6 positive, suggesting their germinal centre origin; one was also CD5 positive. An extranodal presentation occurred in three of five cases. CD56 expression in B cell lymphoma is a rare occurrence. FC is able to identify aberrant immunophenotypes that can be useful in the identification and monitoring of B cell lymphoma subtypes. The presence of CD56 reported by the literature on certain DLBCL with extranodal presentation might be related to mechanisms involved in growth and expansion. © 2012 Blackwell Publishing Limited.

  16. Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test.

    Bowen, Damian E; Whitwell, James H; Lillford, Lucinda; Henderson, Debbie; Kidd, Darren; Mc Garry, Sarah; Pearce, Gareth; Beevers, Carol; Kirkland, David J


    With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(®) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these

  17. Accurate detection of the tumor clone in peripheral T-cell lymphoma biopsies by flow cytometric analysis of TCR-Vβ repertoire.

    Salameire, Dimitri; Solly, Françoise; Fabre, Blandine; Lefebvre, Christine; Chauvet, Martine; Gressin, Rémy; Corront, Bernadette; Ciapa, Agnès; Pernollet, Martine; Plumas, Joël; Macintyre, Elizabeth; Callanan, Mary B; Leroux, Dominique; Jacob, Marie-Christine


    Multiparametric flow cytometry has proven to be a powerful method for detection and immunophenotypic characterization of clonal subsets, particularly in lymphoproliferative disorders of the B-cell lineage. Although in theory promising, this approach has not been comparably fulfilled in mature T-cell malignancies. Specifically, the T-cell receptor-Vβ repertoire analysis in blood can provide strong evidence of clonality, particularly when a single expanded Vß family is detected. The purpose of this study was to determine the relevance of this approach when applied to biopsies, at the site of tumor involvement. To this end, 30 peripheral T-cell lymphoma and 94 control biopsies were prospectively studied. Vβ expansions were commonly detected within CD4+ or CD8+ T cells (97% of peripheral T-cell lymphoma and 54% of non-peripheral T-cell lymphoma cases); thus, not differentiating malignant from reactive processes. Interestingly, we demonstrated that using a standardized evaluation, the detection of a high Vβ expansion was closely associated with diagnosis of peripheral T-cell lymphoma, with remarkable specificity (98%) and sensitivity (90%). This approach also identified eight cases of peripheral T-cell lymphoma that were not detectable by other forms of immunophenotyping. Moreover, focusing Vβ expression analysis to T-cell subsets with aberrant immunophenotypes, we demonstrated that the T-cell clone might be heterogeneous with regard to surface CD7 or CD10 expression (4/11 cases), providing indication on 'phenotypic plasticity'. Finally, among the wide variety of Vβ families, the occurrence of a Vβ17 expansion in five cases was striking. To our knowledge, this is the first report demonstrating the power of T-cell receptor-Vβ repertoire analysis by flow cytometry in biopsies as a basis for peripheral T-cell lymphoma diagnosis and precise T-cell clone identification and characterization.

  18. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting: Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4{sup +} T cells compared

    Ford, A.L.; Goodsall, A.L.; Sedgwick, J.D. [Centenary Institute of Cancer Medicine and Cell Biology, Sydney (Australia)] [and others


    Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4{sup +} cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45{sup low}CD11b/c{sup +} is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45{sup high}CD11b/c{sup +}) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45{sup high}CD11 b/c{sup +} transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4{sup +} myelin basic protein (MBP)-reactive T cells. CD45{sup high}CD11b/c{sup +} CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4{sup +} T cells to proliferate or secrete IL-2. 60 refs., 6 figs., 1 tab.

  19. Innovations in diagnosis and post-therapeutic monitoring of Chagas disease: Simultaneous flow cytometric detection of IgG1 antibodies anti-live amastigote, anti-live trypomastigote, and anti-fixed epimastigote forms of Trypanosoma cruzi.

    Alessio, Glaucia Diniz; Côrtes, Denise Fonseca; Machado de Assis, Girley Francisco; Júnior, Policarpo Ademar Sales; Ferro, Eloisa Amália Vieira; Antonelli, Lis Ribeiro do Valle; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; de Lana, Marta


    This study developed a remarkable methodological innovation (FC-ATE) which enables simultaneous detection of antibodies specific to the three evolutive forms of Trypanosoma cruzi: live amastigote (AMA), live trypomastigote (TRYPO), and fixed epimastigote (EPI) using a differential fluorescence staining as low (AMA), intermediate (TRYPO), and high (EPI). An outstanding performance (100%) was observed in the discrimination of the chagasic (CH) and non-chagasic (NCH) patients. In the applicability of FC-ATE in the diagnosis of Chagas disease, 100% of the CH samples presented positivity in the percentage of positive fluorescent parasites (PPFP) for all the three forms of T. cruzi. Moreover, 94% of the samples of NCH presented negative values of PPFP with AMA and TRYPO, and 88% with EPI. Samples from the NCH group with false-positive results were those belonging to the leishmaniasis patients. Considering the applicability of this technique in post-therapeutic monitoring of Chagas disease, 100% of non-treated (NT) and treated non-cured (TNC) samples were positive with the three T. cruzi evolutive forms, while a percentage of 100% from samples of the treated cured (TC) patients were negative with AMA, 93% with TRYPO and 96% with EPI. The comparison between FC-ATE and two other flow cytometric tests using the same samples of patients NT, TNC and TC showed that the three techniques presented different reactivities, although categorical correlation between the methodologies was observed. Taken together, the results obtained with the novel FC-ATE method have shown an outstanding performance in the diagnosis and post-therapeutic monitoring of Chagas disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. In vitro holding and PLD-repair. Pt. 2. A flow cytometric and electron microscopic analysis of some mammalian cell lines

    Stevenson, A.F.G. (Institute for Basic Research in Developmental Disabilities, Staten Island, NY (USA)); Palackal, T. (State Univ. of New York, Brooklyn, NY (USA). Div. of Radiobiology); Lange, C.S. (Kiel Univ. (Germany, F.R.). Abt. Frauenheilkunde)


    The repair of potentially lethal damage (PLDR) in mammalian cells is expected to be better in quiescent cultures since PLD is supposedly fixed during cycle progression. Plateau phase cultures, therefore, serve as models because of assumed mitotic quiescence. Four established cell lines (V79, CHO, L5178Y and HELA) and one euploid cell strain IMR-90 have been analysed by flow cytometry and electron microscopy to address questions on quiescence in the plateau phase and the effect of holding (induction of quiescence by nutrient privation). In contrast to commonly held views, our results indicate that the quiescent fraction in cultures from transformed cells is exceedingly low (1% or less). Plateau phase cultures of transformed cells are constantly turning over. Euploid cells like the IMR-90 show true quiescence in the plateau phase. Holding causes typical cytopathological changes. These changes have been ultrastructurely characterised. Resistant sub-populations of cells can be selected out under holding-conditions. Such selected cells show completely different radiobiological characteristics, which raise questions on the interpretation of data on PLDR. (orig.).

  1. Final results of a multicenter trial addressing role of CSF flow cytometric analysis in NHL patients at high risk for CNS dissemination.

    Benevolo, Giulia; Stacchini, Alessandra; Spina, Michele; Ferreri, Andrés J M; Arras, Marcella; Bellio, Laura; Botto, Barbara; Bulian, Pietro; Cantonetti, Maria; Depaoli, Lorella; Di Renzo, Nicola; Di Rocco, Alice; Evangelista, Andrea; Franceschetti, Silvia; Godio, Laura; Mannelli, Francesco; Pavone, Vincenzo; Pioltelli, Pietro; Vitolo, Umberto; Pogliani, Enrico M


    This prospective study compared diagnostic and prognostic value of conventional cytologic (CC) examination and flow cytometry (FCM) of baseline samples of cerebrospinal fluid (CSF) in 174 patients with newly diagnosed aggressive non-Hodgkin lymphoma (NHL). FCM detected a neoplastic population in the CSF of 18 of 174 patients (10%), CC only in 7 (4%; P < .001); 11 patients (14%) were discordant (FCM(+)/CC(-)). At a median follow-up of 46 months, there were 64 systemic progressions and 10 CNS relapses, including 2 patients with both systemic and CNS relapses. Two-year progression-free and overall survival were significantly higher in patients with FCM(-) CSF (62% and 72%) compared with those FCM(+) CSF (39% and 50%, respectively), with a 2-year CNS relapse cumulative incidence of 3% (95% confidence interval [CI], 0-7) versus 17% (95% CI, 0-34; P = .004), respectively. The risk of CNS progression was significantly higher in FMC(+)/CC(-) versus FCM(-)/CC(-) patients (hazard ratio = 8.16, 95% CI, 1.45-46). In conclusion, FCM positivity in the CSF of patients with high-risk NHL is associated with a significantly higher CNS relapse risk and poorer outcome. The combination of IV drugs with a higher CNS bioavailability and intrathecal chemotherapy is advisable to prevent CNS relapses in FCM(+) patients.

  2. Clinical performance of human papillomavirus E6, E7 mRNA flow cytometric assay compared to human papillomavirus DNA typing.

    Kottaridi, Christine; Tsiodras, Sotirios; Spathis, Aris; Chranioti, Aikaterini; Pappas, Asimakis; Kassanos, Dimitrios; Panayiotides, Ioannis; Karakitsos, Petros


    To use flow cytometry to screen cervical samples for the overexpression of human papillomavirus (HPV) E6 and E7 mRNA and compare the performance of this assay with an HPV DNA array for the detection of high-grade cervical lesions. Cervical samples were analyzed for HPV DNA by clinical arrays, and the overexpression of E6 and E7 viral oncogenes was monitored using an HPV mRNA detection kit that quantifies the intracellular HPV E6 and E7 mRNA on a cell-by-cell basis. HPV positivity increased with severity of histologic lesions. On the basis of histology-confirmed CIN 2+ cases the specificity of HPV assay was 73.9% (95% CI 66.07, 80.88), whereas it was 39.3% (95% CI 31.85, 47.1) for the DNA assay. The HPV assay provides an early predictor of persistent HPV infection and may improve cervical cancer screening by increasing the specificity of detecting high-grade lesions.

  3. Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.

    Manjanatha, Mugimane G; Bishop, Michelle E; Pearce, Mason G; Kulkarni, Rohan; Lyn-Cook, Lascelles E; Ding, Wei


    Doxorubicin (DOX) is an antineoplastic drug effective against many human malignancies. DOX's clinical efficacy is greatly limited because of severe cardiotoxicity. To evaluate if DOX is genotoxic in the heart, ~7-week-old, male F344 rats were administered intravenously 1, 2, and 3 mg/kg bw DOX at 0, 24, 48, and 69 hr and the Comet assays in heart, liver, kidney, and testis and micronucleus (MN) assay in the peripheral blood (PB) erythrocytes using flow cytometry were conducted. Rats were euthanized at 72 hr and PB was removed for the MN assay and single cells were isolated from multiple tissues for the Comet assays. None of the doses of DOX induced a significant DNA damage in any of the tissues examined by the alkaline Comet assay. Contrastingly, the glycosylase enzymes-modified Comet assay showed a significant dose dependent increase in the oxidative DNA damage in the cardiac tissue (P ≤ 0.05). In the liver, only the top dose induced significant increase in the oxidative DNA damage (P ≤ 0.05). The histopathology showed no severe cardiotoxicity but non-neoplastic lesions were present in both untreated and treated samples. A severe toxicity likely occurred in the bone marrow because no viable reticulocytes could be screened for the MN assay. Gene expression profiling of the heart tissues showed a significant alteration in the expression of 11 DNA damage and repair genes. These results suggest that DOX is genotoxic in the heart and the DNA damage may be induced primarily via the production of reactive oxygen species.

  4. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

    Hidefumi Uchiyama

    Full Text Available Electron paramagnetic resonance (EPR-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH radicals and hydrogen (H atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO, 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO, and phenyl N-t-butylnitrone (PBN. The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and

  5. EPR-Spin Trapping and Flow Cytometric Studies of Free Radicals Generated Using Cold Atmospheric Argon Plasma and X-Ray Irradiation in Aqueous Solutions and Intracellular Milieu.

    Uchiyama, Hidefumi; Zhao, Qing-Li; Hassan, Mariame Ali; Andocs, Gabor; Nojima, Nobuyuki; Takeda, Keigo; Ishikawa, Kenji; Hori, Masaru; Kondo, Takashi


    Electron paramagnetic resonance (EPR)-spin trapping and flow cytometry were used to identify free radicals generated using argon-cold atmospheric plasma (Ar-CAP) in aqueous solutions and intracellularly in comparison with those generated by X-irradiation. Ar-CAP was generated using a high-voltage power supply unit with low-frequency excitation. The characteristics of Ar-CAP were estimated by vacuum UV absorption and emission spectra measurements. Hydroxyl (·OH) radicals and hydrogen (H) atoms in aqueous solutions were identified with the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 3,3,5,5-tetramethyl-1-pyrroline-N-oxide (M4PO), and phenyl N-t-butylnitrone (PBN). The occurrence of Ar-CAP-induced pyrolysis was evaluated using the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) in aqueous solutions of DNA constituents, sodium acetate, and L-alanine. Human lymphoma U937 cells were used to study intracellular oxidative stress using five fluorescent probes with different affinities to a number of reactive species. The analysis and quantification of EPR spectra revealed the formation of enormous amounts of ·OH radicals using Ar-CAP compared with that by X-irradiation. Very small amounts of H atoms were detected whereas nitric oxide was not found. The formation of ·OH radicals depended on the type of rare gas used and the yield correlated inversely with ionization energy in the order of krypton > argon = neon > helium. No pyrolysis radicals were detected in aqueous solutions exposed to Ar-CAP. Intracellularly, ·OH, H2O2, which is the recombination product of ·OH, and OCl- were the most likely formed reactive oxygen species after exposure to Ar-CAP. Intracellularly, there was no practical evidence for the formation of NO whereas very small amounts of superoxides were formed. Despite the superiority of Ar-CAP in forming ·OH radicals, the exposure to X-rays proved more lethal. The mechanism of free radical formation in aqueous solutions and an

  6. Identification of Streptococcus pneumoniae serotype 11E, serovariant 11Av and mixed populations by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR spectroscopy and flow cytometric serotyping assay (FCSA.

    Romina Camilli

    Full Text Available BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new

  7. Flow cytometric analysis of peripheral blood leukemic cells in relapse of acute leukemia%急性白血病复发外周血白血病细胞的流式细胞术检验分析

    杨莉; 何浩明


    Objective To analyse status of peripheral blood leukemic cells detected by flow cytometry in patients with acute leu‐kemia(AL) ,and to provide references for evaluating clinical efficacy and prognosis of AL .Methods The peripheral blood specimens of 87 cases of patients with AL ,including 53 cases of patients with acute myelocytic leukemia and 34 cases of patients with acute lymphoblastic leukemia ,were detected by using flow cytometry ,morphological changes in bone marrow cells were detected ,as well . Results The sensitivity ,specificity and positive predictive value in determination of acute myelocytic leukemia was 95 .6% ,34 .5%and 81 .3% respectively ,and those in acute lymphoblastic leukemia was 87 .3% ,45 .6% and 68 .9% respectively ,statistically signif‐icant differences were found in sensitivity ,specificity and positive predictive value (P<0 .05) .A total of 19 cases with negative mini‐mal residual disease had recurrence(26 .31% ) after 24 months ,and 68 cases with positive minimal residual disease had recurrence (86 .76% ) after 24 months ,and the recurrence rate between the two groups was statistically significant (P<0 .05) .Among all pa‐tients with positive minimal residual disease ,the recurrence rate in patients with high expression level of minimal residual disease (88 .23% ) was higher than that in patients with low expression level of minimal residual disease (47 .09% ) ,and the difference was statistically significant (P<0 .05) .Conclusion Flow cytometric analysis of peripheral blood leukemic cells may has significance for diagnosing relapse of AL and guiding clinical medication .%目的:分析急性白血病(AL)患者外周血白血病细胞流式细胞术检测情况,为分析AL的临床疗效和预后提供参考。方法采用流式细胞术检测87例A L患者(急性髓细胞白血病53例、急性淋巴细胞白血病34例)外周血标本,同时检测骨髓细胞形态学变化。结果急性髓细胞白

  8. Changes of proliferation kinetics after X-irradiation of a human malignant melanoma grown in nude mice

    Spang-Thomsen, M; Vindeløv, L L


    A human malignant melanoma grown in nude mice was exposed to single-dose X-irradiation and the effect on the proliferation kinetics was investigated by two methods. Flow cytometric DNA analysis was performed on tumour tissue obtained by sequential fine-needle aspirations after the treatment to mo......-related increasing proportion of radiation-inactivated tumour cells....

  9. Cytometric Catheter for Neurosurgical Applications

    Evans III, Boyd Mccutchen [ORNL; Allison, Stephen W [ORNL; Fillmore, Helen [ORNL; Broaddus, William C [ORNL; Dyer, Rachel L [ORNL; Gillies, George [ORNL


    Implantation of neural progenitor cells into the central nervous system has attracted strong interest for treatment of a variety of pathologies. For example, the replacement of dopamine-producing (DA) neural cells in the brain appears promising for the treatment of patients affected by Parkinson's disease. Previous studies of cell-replacement strategies have shown that less than 90% of implanted cells survive longer than 24 - 48 hours following the implantation procedure. However, it is unknown if these cells were viable upon delivery, or if they were affected by other factors such as brain pathology or an immune response. An instrumented cell-delivery catheter has been developed to assist in answering these questions by facilitating quantification and monitoring of the viability of the cells delivered. The catheter uses a fiber optic probe to perform flourescence-based cytometric measurments on cells exiting the port at the catheter tip. The current implementation of this design is on a 3.2 mm diameter catheter with 245 micrometer diameter optical fibers. Results of fluorescence testing data are presented and show that the device can characterize the quantity of cell densities ranging from 60,000 cells/ml to 600,000 cells/ml with a coefficient of determination of 0.93.

  10. Local cerebral blood flow and glucose metabolism during seizure in spontaneously epileptic El mice

    Hosokawa, Chisa; Ochi, Hironobu; Yamagami, Sakae; Kawabe, Joji; Kobashi, Toshiko; Okamura, Terue; Yamada, Ryusaku [Osaka City Univ. (Japan). Faculty of Medicine


    Local cerebral blood flow and glucose metabolism were examined in spontaneously epileptic El mice using autoradiography with {sup 125}I-IMP and {sup 14}C-DG in the interictal phase and during seizure. El (+) mice that developed generalized tonic-clonic convulsions and El (-) mice that received no stimulation and had no history of epileptic seizures were examined. The seizure non-susceptible, maternal strain ddY mice were used as control. Uptake ratios for IMP and DG in mouse brain were calculated using the autoradiographic density. In the interictal phase, the pattern of local cerebral blood flow of El (+) mice was similar to that of ddY and El (-) mice, and glucose metabolism in the hippocampus was higher in El (+) mice than in El (-) and ddY mice, but flow and metabolism were nearly matched. During seizure, no significant changed blood flow and increased glucose metabolism in the hippocampus, the epileptic focus, and no markedly changed blood flow and depressed glucose metabolism in other brain regions were observed and considered to be flow-metabolism uncoupling. These observations have never been reported in clinical or experimental studies of epilepsy. Seizures did not cause large regional differences in cerebral blood flow. Therefore, only glucose metabolism is useful for detection of the focus of secondary generalized seizures in El mice, and appeared possibly to be related to the pathophysiology of secondary generalized epilepsy in El mice. (author).

  11. Cytometric patterns reveal growth states of Shewanella putrefaciens.

    Melzer, Susanne; Winter, Gudrun; Jäger, Kathrin; Hübschmann, Thomas; Hause, Gerd; Syrowatka, Frank; Harms, Hauke; Tárnok, Attila; Müller, Susann


    Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  12. Laser speckle contrast imaging of cerebral blood flow of newborn mice at optical clearing

    Timoshina, Polina A.; Zinchenko, Ekaterina M.; Tuchina, Daria K.; Sagatova, Madina M.; Semyachkina-Glushkovskaya, Oxana V.; Tuchin, Valery V.


    In this work, we consider the use of optical clearing agents to improve imaging quality of the cerebral blood flow of newborn mice. Aqueous 60%-glycerol solution, aqueous 70%-OmnipaqueTM(300) solution and OmnipaqueTM (300) solution in water/DMSO(25%/5%) were selected as the optical clearing agents. Laser speckle contrast imaging (LSCI) was used for imaging of cerebral blood flow in newborn mice brain during topical optical clearing of tissuesin the area of the fontanelle. These results demonstrate the effectiveness of glycerol and Omnipaque solutions as optical clearing agents for investigation of cerebral blood flow in newborn mice without scalp removing and skull thinning.

  13. Downregulation of pro-inflammatory cytokines by lupeol measured using cytometric bead array immunoassay.

    Ahmad, Sheikh Fayaz; Pandey, Anjali; Kour, Kiranjeet; Bani, Sarang


    The objective of the study was to investigate the activity of Lupeol (LUP) on proinflammatory and anti-inflammatory cytokines in the pleural exudate from male swiss albino mice. We applied Cytometric bead array technology for simultaneously measurement of these cytokines in pleurisy induced mice treated with lupeol in graded oral doses. Cytometric bead array uses the sensitivity of amplified fluorescence detection by flowcytometer to measure soluble analytes in a particle based immune assay. This assay can accurately quantitate 5 cytokines in a 50 microlitre sample volume. Oral administration of LUP at doses of 25, 50, 100 and 200 mg/kg p.o. produced dose related inhibition of IL-2, IFN-gamma and TNF-alpha in the pleural exudate with the most significant effect at 100 mg/kg oral dose. LUP had a non significant inhibitory effect on the levels of IL-4 and IL-5.

  14. Oral squamous cell carcinoma. Cytometric parameters of prognostic interest.

    Saiz-Bustillo, Ramón; Corchero-Martín, Guadalupe; García-Montesinos-Perea, Belén; Gonzalez-Terán, Tomás; Sánchez-Santolino, Sergio


    The present study was made in order to find possible prognostic factors in oral squamous cell carcinoma, given that it is a frequent disease (3-4% of all malignant tumors) and is the cause of a high morbidity and mortality which justifies any attempt to contribute something towards the understanding of this pathology. 81 oral squamous cell carcinomas, treated with the same procedure, and retrieved from the archive of the Hospital Universitario Marqués de Valdecilla (Santander) were studied. Flow cytometry was carried out on 67 of the samples. No statistically significant differences were found between the cellular proliferative index and the mitotic index, ploidy and the S-phase factor. Likewise, none of the cytometric variables studied presented any association with the appearance of local relapse, distant metastases or survival. These variables cannot be used as a prognostic factors in squamous cell carcinomas of the oral cavity.

  15. Chronic Stress Impairs Collateral Blood Flow Recovery in Aged Mice


    0, normal; 1–5, cyanosis or loss of nail(s), where the score is dependent on the number of nails affected; 6–10, partial or complete atrophy of...digit(s), where the score reflects the number of digits affected; and 11, partial atrophy of forefoot [21]. Hind limb use scores (index of muscle function...brain, and cardiac fibrosis and, in many ways, provides a posttraumatic stress disorder (PTSD) model for mice [26]. As a model of physical/neurogenic

  16. A Triple-Stain Flow Cytometric Method to Assess Plasma- and Acrosome-Membrane Integrity of Cryopreserved Bovine Sperm Immediately after Thawing in Presence of Egg-Yolk Particles

    Nagy, S.; Jansen, J.; Topper, E.K.; Gadella, B.M.


    Simultaneously evaluating postthaw viability and acrosome integrity of spermatozoa by flow cytometry would provide a valuable testing tool in both research and routine work. In the present study, a new triple-stain combination was developed for the simultaneous evaluation of viability and acrosome i

  17. Multicentric study underlining the interest of adding CD5, CD7 and CD56 expression assessment to the flow cytometric Ogata score in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

    Bardet, Valérie; Wagner-Ballon, Orianne; Guy, Julien; Morvan, Céline; Debord, Camille; Trimoreau, Franck; Benayoun, Emmanuel; Chapuis, Nicolas; Freynet, Nicolas; Rossi, Cédric; Mathis, Stéphanie; Gourin, Marie-Pierre; Toma, Andréa; Béné, Marie C; Feuillard, Jean; Guérin, Estelle


    Although numerous recent publications have demonstrated interest in multiparameter flow cytometry in the investigation of myelodysplastic disorders, it is perceived by many laboratory hematologists as difficult and expensive, requiring a high level of expertise. We report a multicentric open real-life study aimed at evaluating the added value of the technically simple flow cytometry score described by the Ogata group for the diagnosis of myelodysplastic syndromes. A total of 652 patients were recruited prospectively in four different centers: 346 myelodysplastic syndromes, 53 myelodysplastic/myeloproliferative neoplasms, and 253 controls. The Ogata score was assessed using CD45 and CD34 staining, with the addition of CD10 and CD19. Moreover, labeling of CD5, CD7 and CD56 for the evaluation of myeloid progenitors and monocytes was tested on a subset of 294 patients. On the whole series, the specificity of Ogata score reached 89%. Respective sensitivities were 54% for low-risk myelodysplastic syndromes, 68% and 84% for type 1 and type 2 refractory anemia with excess of blasts, and 72% for myelodysplastic/myeloproliferative neoplasms. CD5 expression was poorly informative. When adding CD56 or CD7 labeling to the Ogata score, sensitivity rose to 66% for low-risk myelodysplastic syndromes, to 89% for myelodysplastic/myeloproliferative neoplasms and to 97% for refractory anemia with excess of blasts. This large multicenter study confirms the feasibility of Ogata scoring in routine flow cytometry diagnosis but highlights its poor sensitivity in low-risk myelodysplastic syndromes. The addition of CD7 and CD56 in flow cytometry panels improves the sensitivity but more sophisticated panels would be more informative. Copyright© Ferrata Storti Foundation.

  18. Flow cytometric detection of micronuclei and cell cycle alterations in fish-derived cells after exposure to three model genotoxic agents: mitomycin C, vincristine sulfate and benzo(a)pyrene.

    Sánchez, P; Llorente, M T; Castaño, A


    The measurement of cytogenetic alterations in vitro is considered an initial step in the risk assessment procedures for genotoxic agents. The concern about genotoxic pollutants in natural fish population makes the use of fish-derived cells an useful tool for these purposes. The technological improvements in well-established cytogenetic endpoints, such as micronuclei (MN) estimations by means of flow cytometry, have been proposed in the later years using mammalian cells. In this work, we test the capability of flow cytometry to evaluate MN induction and cell cycle alterations in an established fish cell line (RTG-2) using three agent-inductor models at different concentrations and exposure periods. For mitomycin C, an inverse relationship between length of exposure period and concentrations was observed. A dose-response relationship was observed after exposing RTG-2 cells to vincristine sulfate and benzo(a)pyrene. As this study shows, RTG-2 cells respond to clastogenic and aneugenic effects of the tested chemicals through the induction of MN at similar doses to mammalian cells and without the addition of exogenous metabolic activity. The possibility to check cell cycle alterations, in the same sample, gives the opportunity to evaluate early signals of cytotoxicity. The use of flow cytometry improves the assay by means of its speed and objectivity, which makes the assay very useful for genotoxicity assessment of aquatic chemicals.

  19. Immune complex stimulation of human neutrophils involves a novel Ca2+/H+ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca2+/pH responses by subpopulations.

    Bernardo, John; Hartlaub, Hilary; Yu, Xin; Long, Heidi; Simons, Elizabeth R


    The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described. The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca(2)(+)/H(+) voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

  20. Flow-cytometric method for observing the effects of pulsed electromagnetic fields on the growth of rat bone marrow mesenchymal stem cells%流式细胞仪观察脉冲电磁场干预骨髓间充质干细胞的生长

    黄钊; 苏伟; 崔向荣; 覃万安


    BACKGROUND: Compared with the morphology, DNA electrophoresis and other methods, flow-cytometric method has more advantages on the detection of cell phenotype, proliferation rate and cell cycle of rat bone marrow mesenchymal stem cells (BMSCs). OBJECTIVE: To observe and discuss the effect of specific pulsed electromagnetic fields stimulation on the proliferation and cell cycle of rat BMSCs with the flow-cytometric method (FCM). METHODS: Rat BMSCs were exposed to pulsed electromagnetic fields (frequency for 1 kHz, magnetic flux density for 0.05 mT, power density for 5 mW/cm2). BMSCs without exposure to pulsed electromagnetic fields were used as controls. Expression of CD29, CD31, CD44 CD45, CD105 were detect in the control group. The proliferation rate and cell cycle of passage 3 cells were detected at 3, 6, 9, 12 days. RESULTS AND CONCLUSION: CD29, CD44, CD105 in the 3rd passage non-stimulated BMSCs was positively expressed and the CD31, CD45 was negatively expressed (P < 0.05). The survival rate of passage 3 BMSCs and percentage of S phase following intervention of pulsed electromagnetic fields were greater than those in the control group (P < 0.05). These results indicated that the specific pulsed electromagnetic fields can promote the growth and proliferation of BMSCs to some extent.%背景:应用流式细胞仪检测细胞表型、存活或凋亡细胞计数及细胞周期,较形态学观察、DNA电泳等检测方法更具优势.目的:采用流式细胞仪分选检测特定脉冲电磁场干预对大鼠骨髓间充质干细胞生长周期及其增殖效率的影响.方法:使用振荡频率1 kHz,磁感应强度0.05 mT、功率密度5 mW/cm2的脉冲电磁场照射大鼠骨髓间充质干细胞,以未经磁场干预的细胞为对照.采用流式细胞仪检测未经磁场干预细胞表面抗原CD29、CD31、CD44、CD45和CD105表达率;于传第3代后第3,6,9,12天测定不同干预细胞增殖率及生长周期.结果与结论:未经磁场干预的第3代

  1. Flow cytometric analysis of mitotic cycle perturbation by chemical carcinogens in cultured epithelial cells. [Effects of benzo(a)pyrene-diol-epoxide on mitotic cycle of cultural mouse liver epithelial cells

    Pearlman, A.L.


    A system for kinetic analysis of mitotic cycle perturbation by various agents was developed and applied to the study of the mitotic cycle effects and dependency of the chemical carcinogen benzo(a)pyrene-diolepoxide, DE, upon a mouse lever epithelial cell line, NMuLi. The study suggests that the targets of DE action are not confined to DNA alone but may include cytoplasmic structures as well. DE was found to affect cells located in virtually every phase of the mitotic cycle, with cells that were actively synthesizing DNA showing the strongest response. However, the resulting perturbations were not confined to S-phase alone. DE slowed traversal through S-phase by about 40% regardless of the cycle phase of the cells exposed to it, and slowed traversal through G/sub 2/M by about 50%. When added to G/sub 1/ cells, DE delayed recruitment of apparently quiescent (G/sub 0/) cells by 2 hours, and reduced the synchrony of the cohort of cells recruited into active proliferation. The kinetic analysis system consists of four elements: tissue culture methods for propagating and harvesting cell populations; an elutriation centrifugation system for bulk synchronization of cells in various phases of the mitotic cycle; a flow cytometer (FCM), coupled with appropriate staining protocols, to enable rapid analysis of the DNA distribution of any given cell population; and data reduction and analysis methods for extracting information from the DNA histograms produced by the FCM. The elements of the system are discussed. A mathematical analysis of DNA histograms obtained by FCM is presented. The analysis leads to the detailed implementation of a new modeling approach. The new modeling approach is applied to the estimation of cell cycle kinetic parameters from time series of DNA histograms, and methods for the reduction and interpretation of such series are suggested.

  2. Intestinal intraepithelial lymphocyte cytometric pattern is more accurate than subepithelial deposits of anti-tissue transglutaminase IgA for the diagnosis of celiac disease in lymphocytic enteritis.

    Fernando Fernández-Bañares

    Full Text Available BACKGROUND & AIMS: An increase in CD3+TCRγδ+ and a decrease in CD3- intraepithelial lymphocytes (IEL is a characteristic flow cytometric pattern of celiac disease (CD with atrophy. The aim was to evaluate the usefulness of both CD IEL cytometric pattern and anti-TG2 IgA subepithelial deposit analysis (CD IF pattern for diagnosing lymphocytic enteritis due to CD. METHODS: Two-hundred and five patients (144 females who underwent duodenal biopsy for clinical suspicion of CD and positive celiac genetics were prospectively included. Fifty had villous atrophy, 70 lymphocytic enteritis, and 85 normal histology. Eight patients with non-celiac atrophy and 15 with lymphocytic enteritis secondary to Helicobacter pylori acted as control group. Duodenal biopsies were obtained to assess both CD IEL flow cytometric (complete or incomplete and IF patterns. RESULTS: Sensitivity of IF, and complete and incomplete cytometric patterns for CD diagnosis in patients with positive serology (Marsh 1+3 was 92%, 85 and 97% respectively, but only the complete cytometric pattern had 100% specificity. Twelve seropositive and 8 seronegative Marsh 1 patients had a CD diagnosis at inclusion or after gluten free-diet, respectively. CD cytometric pattern showed a better diagnostic performance than both IF pattern and serology for CD diagnosis in lymphocytic enteritis at baseline (95% vs 60% vs 60%, p = 0.039. CONCLUSIONS: Analysis of the IEL flow cytometric pattern is a fast, accurate method for identifying CD in the initial diagnostic biopsy of patients presenting with lymphocytic enteritis, even in seronegative patients, and seems to be better than anti-TG2 intestinal deposits.

  3. 应用流式微球检测黄曲霉毒素B1方法的建立%Determination of aflatoxin B1 based on a flow cytometric microsphere immunoassay

    李泳宁; 吴海燕; 郑允权; 郭养浩


    采用活性酯法,将AFB1-BSA人工抗原交联于含有羧基表面的荧光微球,通过与游离AFB1竞争抗AFB1单克隆抗体后,再与异硫氰酸荧光素标记二抗的反应,建立基于微球的间接竞争免疫检测方法.检测结果表明,流式细胞仪检测AFB1的检测限为0.03 ng·mL-1,检测范围为0.05 ~ 1.0 ng· mL-1.与黄曲霉毒素B2、黄曲霉毒素G1、黄曲霉毒素G2、黄曲霉毒素M1和黄曲霉毒素M2的交叉反应率均低于1.0%.在玉米样品中的加标回收率为87% ~ 103%,变异系数为6.1%~8.4%.%Carboxyl - modified microspheres internally dyed with fluroscent dye were conjugated with the artificial antigen AFB, - BSA. Aflatoxin B, ( AFB,) was used as a positive control to compete with the AFB, - BSA antigen on the surface of the microspheres for the anti - AFB, McAb. A fluorescein isothiocyanate labeled IgG reporter antibody was added to react specifically with the anti - AFB, McAb on the microspheres. The detection limit of AFB, reached 0.03 ng · mL-1, with a good linearity ranging 0.05 ~ 1.0 ng mL-1. The cross - reactivity rates were less than 1.0% with other toxins such as aflatoxins B2, aflatoxins G,, aflatoxins G2, aflatoxins M, and aflatoxins M2. The recovery of AFB, from artificially contaminated corn samples was from 89% to 92% , with CVs from 6.8% to 9.0%. A novel method for the determination of aflatoxin B, by an indirect competitive immunoassay with a flow cytometer has been developed.

  4. Analysis of the Fine-Scale Population Structure of “Candidatus Accumulibacter phosphatis” in Enhanced Biological Phosphorus Removal Sludge, Using Fluorescence In Situ Hybridization and Flow Cytometric Sorting▿

    Kim, Jeong Myeong; Lee, Hyo Jung; Kim, Sun Young; Song, Jae Jun; Park, Woojun; Jeon, Che Ok


    To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “Candidatus Accumulibacter phosphatis” (henceforth referred to as “Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “Ca. Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 (ppk1) gene homologs were amplified by using a ppk1-specific PCR primer set for “Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes

  5. Noninvasive optical measurement of cerebral blood flow in mice using molecular dynamics analysis of indocyanine green.

    Taeyun Ku

    Full Text Available In preclinical studies of ischemic brain disorders, it is crucial to measure cerebral blood flow (CBF; however, this requires radiological techniques with heavy instrumentation or invasive procedures. Here, we propose a noninvasive and easy-to-use optical imaging technique for measuring CBF in experimental small animals. Mice were injected with indocyanine green (ICG via tail-vein catheterization. Time-series near-infrared fluorescence signals excited by 760 nm light-emitting diodes were imaged overhead by a charge-coupled device coupled with an 830 nm bandpass-filter. We calculated four CBF parameters including arrival time, rising time and mean transit time of a bolus and blood flow index based on time and intensity information of ICG fluorescence dynamics. CBF maps were generated using the parameters to estimate the status of CBF, and they dominantly represented intracerebral blood flows in mice even in the presence of an intact skull and scalp. We demonstrated that this noninvasive optical imaging technique successfully detected reduced local CBF during middle cerebral artery occlusion. We further showed that the proposed method is sufficiently sensitive to detect the differences between CBF status in mice anesthetized with either isoflurane or ketamine-xylazine, and monitor the dynamic changes in CBF after reperfusion during transient middle cerebral artery occlusion. The near-infrared optical imaging of ICG fluorescence combined with a time-series analysis of the molecular dynamics can be a useful noninvasive tool for preclinical studies of brain ischemia.

  6. Flow Cytometric Evidence for Hydroxyl Radical-induced Apoptosis in Tobacco Protoplasts%羟自由基诱导的烟草原生质体的凋亡:流式细胞法的新证据

    雷晓勇; 廖旭东; 张贵友; 戴尧仁


    用1.0 mmol/L FeSO4/0.5 mmol/L H202处理烟草(Nicotiana tabacum L.cultivar BY 2)原生质体,发现羟自由基能够诱导烟草原生质体的凋亡.具体表现为细胞核皱缩、DNA Ladder、TUNEL阳性反应等典型的凋亡特征.在动物细胞凋亡过程中,线粒体起着非常重要的作用,其中膜电位(△ψm)的变化以及由其引起的位于线粒体膜上的通透性孔(PTP)的开放与Cyt c的释放有关.另外,在动物凋亡细胞中,磷脂酰丝氨酸(phosphatidyl serine,PS)会从细胞膜内侧向外翻转.为了判断植物细胞凋亡过程中膜电位的变化情况以及PS的外翻程度,我们采用了流式细胞法.结果表明,随着处理时间的延长,烟草原生质体线粒体的膜电位逐渐降低;膜内PS大量外翻.说明由羟自由基和烟草原生质体组成的凋亡体系是一种可靠的凋亡组合,可以用来对植物细胞凋亡机理做进一步研究.%Protoplasts prepared from tobacco (Nicotiana tabacum L., cultivar BY-2) suspension cellshave similar morphological characteristics to those in animal cells. The hallmarks of apoptosis such ascondensation and peripheral distribution of nuclei, TUNEL positive reaction, and DNA ladders were ob-served when tobacco protoplasts were treated with the hydroxyl radical generating system (1.0 mmol/LFeSO4/0.5 mmol/L H2O2). In animals, the loss of transmembrane potential (△ψm ) and the exposure ofphospholipid phosphatidylserine (PS) are believed to be the main apoptosis events. To test whether thesesignificant processes take place in plants, flow cytometry was used to detect annexin V binding andchanges in △ψm. Results showed that the PS turned out from inner membrane and △ψm graduallydecreased during the apoptosis. All these apoptotic characteristics proved that hydroxyl radicals cancause typical programmed cell death (PCD) in tobacco protoplasts and this design can be served as aneffective experiment system to explore the mechanism of plant apoptosis.

  7. Ager Deletion Enhances Ischemic Muscle Inflammation, Angiogenesis, and Blood Flow Recovery in Diabetic Mice.

    López-Díez, Raquel; Shen, Xiaoping; Daffu, Gurdip; Khursheed, Md; Hu, Jiyuan; Song, Fei; Rosario, Rosa; Xu, Yunlu; Li, Qing; Xi, Xiangmei; Zou, Yu Shan; Li, Huilin; Schmidt, Ann Marie; Yan, Shi Fang


    Diabetic subjects are at higher risk of ischemic peripheral vascular disease. We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block angiogenesis and blood flow recovery after hindlimb ischemia induced by femoral artery ligation through modulation of immune/inflammatory mechanisms. Wild-type mice rendered diabetic with streptozotocin and subjected to unilateral femoral artery ligation displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic wild-type mice, femoral artery ligation attenuated angiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including Ccl2 (chemokine [C-C motif] ligand-2) and Egr1 (early growth response gene-1) versus nondiabetic mice. Deletion of Ager (gene encoding RAGE) or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, angiogenesis, and blood flow recovery in diabetic mice. In diabetes mellitus, deletion of Ager increased circulating Ly6C(hi) monocytes and augmented macrophage infiltration into ischemic muscle tissue after femoral artery ligation. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. These findings indicate that RAGE attenuates adaptive inflammation in hindlimb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic peripheral vascular disease. © 2017 American Heart Association, Inc.

  8. Seasonality in molecular and cytometric diversity of marine bacterioplankton: the reshuffling of bacterial taxa by vertical mixing

    García, Francisca C.


    The ’cytometric diversity’ of phytoplankton communities has been studied based on single-cell properties, but the applicability of this method to characterize bacterioplankton has been unexplored. Here, we analysed seasonal changes in cytometric diversity of marine bacterioplankton along a decadal time-series at three coastal stations in the Southern Bay of Biscay. Shannon-Weaver diversity estimates and Bray-Curtis similarities obtained by cytometric and molecular (16S rRNA tag sequencing) methods were significantly correlated in samples from a 3.5-year monthly time-series. Both methods showed a consistent cyclical pattern in the diversity of surface bacterial communities with maximal values in winter. The analysis of the highly resolved flow cytometry time-series across the vertical profile showed that water column mixing was a key factor explaining the seasonal changes in bacterial composition and the winter increase in bacterial diversity in coastal surface waters. Due to its low cost and short processing time as compared to genetic methods, the cytometric diversity approach represents a useful complementary tool in the macroecology of aquatic microbes.

  9. Flow cytometric detection of viruses in the Zuari estuary, Goa

    Mitbavkar, S.; Rajaneesh, K.M.; SathishKumar, P.

    and microalgae, the most abundant organisms in the ocean and also micro- zooplankton 2 . They have been implicated in phytoplankton mortality and the de- cline of phytoplankton blooms 1 . Marine phytoplankton is responsible for up to half of the total primary...

  10. Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients

    Warzynski, Michael J; Graham, David M; Axtell, Richard A; Higgins, James V; Hammers, Yuki A


    .... Following the “rare event” philosophy of selecting one negative and two positive antigens, we initially tried a “cocktail” of CD45 − CD56 very bright+ neuron‐specific enolase (NSE) cytoplasmic...

  11. Flow Cytometric Ploidy Determination of Oral Premalignant and Malignant Lesions


    However, subjectivity remains an inherent part of the diagnostic process.9 According to Dabelsteen,10 subjectivity is most apparent in the determination...of Silverman 16 and Pindborg43 and Mincer et al.21 that light microscopio features of premalignancy are often not present in original biopsy specimens...was caused either in whole or in part by the presence of doublets was eliminated by syringina,. filtering and visually examining the population. In

  12. Flow Cytometric Applicability of Fluorescent Vitality Probes on Phytoplankton

    Peperzak, L.; Brussaard, C.P.D.


    The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein-AM], 5-chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2',7'-dichlorofluorescein diacetate [H(2)DCFDA]; and two membrane probes: bis-(1,3-dibutylbarbituric acid) tri

  13. Cytometric Approach for Detection of Encephalitozoon intestinalis, an Emergent Agent▿

    Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pina-Vaz, Cidália


    Encephalitozoon intestinalis is responsible for intestinal disease in patients with AIDS and immunocompetent patients. The infectious form is a small spore that is resistant to water treatment procedures. Its detection is very important, but detection is very cumbersome and time-consuming. Our main objective was to develop and optimize a specific flow cytometric (FC) protocol for the detection of E. intestinalis in hospital tap water and human feces. To determine the optimal specific antibody (Microspor-FA) concentration, a known concentration of E. intestinalis spores (Waterborne, Inc.) was suspended in hospital tap water and stool specimens with different concentrations of Microspor-FA, and the tap water and stool specimens were incubated under different conditions. The sensitivity limit and specificity were also evaluated. To study spore infectivity, double staining with propidium iodide (PI) and Microspor-FA was undertaken. Distinct approaches for filtration and centrifugation of the stool specimens were used. E. intestinalis spores stained with 10 μg/ml of Microspor-FA at 25°C overnight provided the best results. The detection limit was 5 × 104 spores/ml, and good specificity was demonstrated. Simultaneous staining with Microspor-FA and PI ensured that the E. intestinalis spores were dead and therefore noninfectious. With the stool specimens, better spore recovery was observed with a saturated solution of NaCl and centrifugation at 1,500 × g for 15 min. A new approach for the detection of E. intestinalis from tap water or human feces that ensures that the spores are not viable is now available and represents an important step for the prevention of this threat to public health. PMID:19439525

  14. Differentially Severe Cognitive Effects of Compromised Cerebral Blood Flow in Aged Mice: Association with Myelin Degradation and Microglia Activation

    Gilly Wolf


    Full Text Available Bilateral common carotid artery stenosis (BCAS models the effects of compromised cerebral blood flow on brain structure and function in mice. We compared the effects of BCAS in aged (21 month and young adult (3 month female mice, anticipating a differentially more severe effect in the older mice. Four weeks after surgery there was a significant age by time by treatment interaction on the radial-arm water maze (RAWM; p = 0.014: on the first day of the test, latencies of old mice were longer compared to the latencies of young adult mice, independent of BCAS. However, on the second day of the test, latencies of old BCAS mice were significantly longer than old control mice (p = 0.049, while latencies of old controls were similar to those of the young adult mice, indicating more severe impairment of hippocampal dependent learning and working memory by BCAS in the older mice. Fluorescence staining of myelin basic protein (MBP showed that old age and BCAS both induced a significant decrease in fluorescence intensity. Evaluation of the number oligodendrocyte precursor cells demonstrated augmented myelin replacement in old BCAS mice (p < 0.05 compared with young adult BCAS and old control mice. While microglia morphology was assessed as normal in young adult control and young adult BCAS mice, microglia of old BCAS mice exhibited striking activation in the area of degraded myelin compared to young adult BCAS (p < 0.01 and old control mice (p < 0.05. These findings show a differentially more severe effect of cerebral hypoperfusion on cognitive function, myelin integrity and inflammatory processes in aged mice. Hypoperfusion may exacerbate degradation initiated by aging, which may induce more severe neuronal and cognitive phenotypes.

  15. Psychological stress impairs hepatic blood flow via central CRF receptors in mice.

    Chida, Yoichi; Sudo, Nobuyuki; Kubo, Chiharu


    Some previous works have further expanded the 'Brain-Gut axis', that is a bi-directional interaction between the gut and brain function, into a relationship of the brain with the liver. However, all the details of such brain-liver relationship were not fully understood because appropriate animal models had not been established yet. Here we developed a novel animal model, in which hepatic blood flow of conscious mice could be measured in real-time. In addition, using this model, we also demonstrated that exposure to psychological stress considerably reduced hepatic blood flow via central CRF receptors. Thus, this new model is considered to be a useful and promising tool for elucidating the precise effects of emotional factors on liver function.

  16. Effects of anesthesia on the cerebral capillary blood flow in young and old mice

    Moeini, Mohammad; Tabatabaei, Maryam S.; Bélanger, Samuel; Avti, Pramod; Castonguay, Alexandre; Pouliot, Philippe; Lesage, Frédéric


    Despite recent findings on the possible role of age-related cerebral microvasculature changes in cognition decline, previous studies of capillary blood flow in aging (using animal models) are scarce and limited to anesthetized conditions. Since anesthesia can have different effects in young and old animals, it may introduce a confounding effect in aging studies. The present study aimed to eliminate the potential confound introduced by anesthesia by measuring capillary blood flow parameters in both awake conditions and under isoflurane anesthesia. We used 2-photon laser scanning fluorescence microscopy to measure capillary diameter, red blood cell velocity and flux, hematocrit and capillary volumetric flow in individual capillaries in the barrel cortex of 6- and 24-month old C57Bl/6 mice. It was observed that microvascular properties are significantly affected by anesthesia leading to different trends in capillary blood flow parameters with aging when measured under awake or anesthetized conditions. The findings in this study suggest taking extra care in interpreting aging studies from anesthetized animals.

  17. Electromagnetic treatment to old Alzheimer's mice reverses β-amyloid deposition, modifies cerebral blood flow, and provides selected cognitive benefit.

    Arendash, Gary W; Mori, Takashi; Dorsey, Maggie; Gonzalez, Rich; Tajiri, Naoki; Borlongan, Cesar


    Few studies have investigated physiologic and cognitive effects of "long-term" electromagnetic field (EMF) exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25-1.05 W/kg) by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer's transgenic (Tg) mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain β-amyloid (Aβ) aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21-27 month) Tg mice over a 2-month period reverses their very advanced brain Aβ aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature) during EMF "ON" periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated Aβ (Tg mice) and slight body hyperthermia during "ON" periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer's Tg mice and normal mice, as well as reversal of advanced Aβ neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF treatment

  18. Electromagnetic treatment to old Alzheimer's mice reverses β-amyloid deposition, modifies cerebral blood flow, and provides selected cognitive benefit.

    Gary W Arendash

    Full Text Available Few studies have investigated physiologic and cognitive effects of "long-term" electromagnetic field (EMF exposure in humans or animals. Our recent studies have provided initial insight into the long-term impact of adulthood EMF exposure (GSM, pulsed/modulated, 918 MHz, 0.25-1.05 W/kg by showing 6+ months of daily EMF treatment protects against or reverses cognitive impairment in Alzheimer's transgenic (Tg mice, while even having cognitive benefit to normal mice. Mechanistically, EMF-induced cognitive benefits involve suppression of brain β-amyloid (Aβ aggregation/deposition in Tg mice and brain mitochondrial enhancement in both Tg and normal mice. The present study extends this work by showing that daily EMF treatment given to very old (21-27 month Tg mice over a 2-month period reverses their very advanced brain Aβ aggregation/deposition. These very old Tg mice and their normal littermates together showed an increase in general memory function in the Y-maze task, although not in more complex tasks. Measurement of both body and brain temperature at intervals during the 2-month EMF treatment, as well as in a separate group of Tg mice during a 12-day treatment period, revealed no appreciable increases in brain temperature (and no/slight increases in body temperature during EMF "ON" periods. Thus, the neuropathologic/cognitive benefits of EMF treatment occur without brain hyperthermia. Finally, regional cerebral blood flow in cerebral cortex was determined to be reduced in both Tg and normal mice after 2 months of EMF treatment, most probably through cerebrovascular constriction induced by freed/disaggregated Aβ (Tg mice and slight body hyperthermia during "ON" periods. These results demonstrate that long-term EMF treatment can provide general cognitive benefit to very old Alzheimer's Tg mice and normal mice, as well as reversal of advanced Aβ neuropathology in Tg mice without brain heating. Results further underscore the potential for EMF

  19. Flow: Statistics, visualization and informatics for flow cytometry

    Kepler Thomas B


    Full Text Available Abstract Flow is an open source software application for clinical and experimental researchers to perform exploratory data analysis, clustering and annotation of flow cytometric data. Flow is an extensible system that offers the ease of use commonly found in commercial flow cytometry software packages and the statistical power of academic packages like the R BioConductor project.

  20. Soluble epoxide hydrolase gene deletion improves blood flow and reduces infarct size after cerebral ischemia in reproductively senescent female mice

    Kristen L Zuloaga


    Full Text Available Soluble epoxide hydrolase (sEH, a key enzyme in the metabolism of vasodilatory epoxyeicosatrienoic acids (EETs, is sexually dimorphic, suppressed by estrogen, and contributes to underlying sex differences in cerebral blood flow and injury after cerebral ischemia. We tested the hypothesis that sEH inhibition or gene deletion in reproductively senescent (RS female mice would increase cerebral perfusion and decrease infarct size following stroke. RS (15-18 month old and young (3-4 month old female sEH knockout (sEHKO mice and wild type (WT mice were subjected to 45 min middle cerebral artery occlusion (MCAO with laser Doppler perfusion monitoring. WT mice were treated with vehicle or a sEH inhibitor t-AUCB at the time of reperfusion and every 24hrs thereafter for 3 days. Differences in regional cerebral blood flow were measured in vivo using optical microangiography. Infarct size was measured 3 days after reperfusion. Infarct size and cerebral perfusion 24h after MCAO were not altered by age. Both sEH gene deletion and sEH inhibition increased cortical perfusion 24h after MCAO. Neither sEH gene deletion nor sEH inhibition reduced infarct size in young mice. However, sEH gene deletion, but not sEH inhibition of the hydrolase domain of the enzyme, decreased infarct size in RS mice. Results of these studies show that sEH gene deletion and sEH inhibition enhance cortical perfusion following MCAO and sEH gene deletion reduces damage after ischemia in RS female mice; however this neuroprotection in absent is young mice.

  1. Flow cytometric monitoring of minimal residual diseases in patients with acute leukemia after allogeneic hemapoietic stem cell transplantation%急性白血病异基因造血干细胞移植后流式细胞术监测微量残留病的意义

    高雁群; 孙媛; 张耀臣; 纪树荃; 陆道培; 吴彤; 王卉; 童春容; 张维婕; 王静波; 卢岳; 赵艳丽; 周葭蕤


    目的 研究急性白血病异基因造血干细胞移植(allo-HSCT)后采用流式细胞术(FCM)监测微量残留病(MRD)的意义.方法 自2007年1月至2008年1月采用FCM对102例初诊时未检测出白血病基因和染色体改变的急性白血病allo-HSCT后患者进行骨髓MRD检测(移植后1、2、3、6、12个月,部分高危患者增加检测频率),观察MRD结果与临床转归的关系,对有意义的MRD增高患者予以临床干预并采用FCM监测疗效.MRD> 0.01%为阳性.结果 ①移植后MRD持续阴性者71例,均为血液学完全缓解(CR),仅3例髓外复发,其无病生存( DFS)及总生存(0S)率分别为66.2%及90.1%.②移植后MRD阳性者27例,经过干预治疗(化疗加供者淋巴细胞输注、多种细胞因子诱导的杀伤细胞和NK细胞治疗),11例患者转阴,其DFS及OS率分别为63.6%及72.7%.另外16例血液学复发,其DFS及OS率分别为11.1%及25.0%.从MRD增高至血液学复发的中位时间为48(7~69)d.③移植后直接血液学复发者共4例,均死亡.结论 移植后采用FCM检测MRD:①MRD持续阴性组患者其DFS及OS率均明显高于MRD阳性组.②移植后出现MRD阳性的患者,通过干预性治疗,MRD再次转阴后,其DFS及OS率仍然高于持续阳性组.③移植后直接血液学复发的患者,其DFS及OS率极低,预后极差.采用FCM监测急性白血病allo-HSCT后MRD是一种敏感、特异、快速、简便的方法,可及时提示复发倾向,便于早期干预治疗,降低血液学复发风险,提高allo-HSCT后患者的DFS率.%Objective To study the significance of flow cytometric monitoring minimal residual diseases (MRD) in patients with acute leukemia (AL) after allogeneic hemapoietic stem cell transplantation (HSCT).Methods From January 2007 and January 2008 MRD were detected by flow cytometry (FCM) in 402 bone marrow (BM) in 102 AL patients without leukemic gene and chromosomal changes at first diagnosis after HSCT( 1,2,3,6,12 months after HSCT

  2. Dietary gluten reduces the number of intestinal regulatory T cells in mice

    Ejsing-Duun, Maria; Josephsen, Jytte; Aasted, Bent


    It is well established that gluten-free diet reduces the incidence of type 1 diabetes mellitus (T1D) in non-obese diabetic (NOD) mice, though the mechanism is not known. However, regulatory T cells (Treg) are likely to play an important role. Also, it is known that dietary gluten induces...... an intestinal increase in the bacterium Lactococcus garvieae, but the importance of this phenomenon for T1D development is doubtful. Our hypothesis is that gluten is responsible for mediating its effect on T1D through the influence on Treg development independent of gluten-induced Lactococci. Four groups...... of female NOD and BALB / c mice of 3 week old were fed either a gluten-free diet or a standard diet. Lactococcus garvieae or saline water was administered per oral to one of each dietary group. Spleen and Peyer's patches were sampled from BALB / c mice for flow cytometric monitoring of IL-10 and Treg. NOD...

  3. Dynamics of testicular germ cell apoptosis in normal mice and transgenic mice overexpressing rat androgen-binding protein

    Petrusz Peter


    Full Text Available Abstract The number and type of testicular germ cells undergoing apoptosis in different age groups of mice (from 7 to 360 days of age was determined and compared in age-matched wild type (WT control and in a transgenic (TG mice homozygous to rat androgen binding protein (ABP using flow cytometry. Flow cytometric quantification revealed that the total number of germ cells undergoing apoptosis did not differ significantly in WT and TG mice up to Day 14. From Day 21 to Day 60, the number of germ cells undergoing apoptosis was consistently higher in TG than in WT mice. Starting from Day 90, the number of germ cells undergoing apoptosis in TG mice was lower than controls until Day 360. In 21–60 days old TG mice, spermatogonia, S-Phase cells, and primary spermatocytes are the cell types undergoing apoptosis at significantly greater numbers than those in WT mice. However, starting from day 60, the total number of spermatids undergoing apoptosis was significantly lower in TG mice than in age-matched WT controls. TdT-mediated dUTP-biotin nick end labeling (TUNEL in testicular sections from TG mice of 21 and 30 days of age confirmed the presence of increased numbers of apoptotic germ cells compared to their age matched controls. These data indicate that the continuous presence of greater than physiological concentrations of ABP in the mouse testis has a biphasic effect on the frequency of apoptosis in germ cells. The initial pre-pubertal increase in testicular germ cell apoptosis may result from direct or indirect actions of ABP and is likely to determine the subsequent life-death balance of germ cell populations in TG mice, whereas the subsequent reduction may result from maturation depletion. A wave of apoptosis during the pre-pubertal period is required for normal spermatogenesis to develop, and our data indicate that this apoptotic wave may be regulated by ABP and/or androgens.

  4. A Dietary Treatment Improves Cerebral Blood Flow and Brain Connectivity in Aging apoE4 Mice

    Maximilian Wiesmann


    Full Text Available APOE ε4 (apoE4 polymorphism is the main genetic determinant of sporadic Alzheimer’s disease (AD. A dietary approach (Fortasyn including docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium has been proposed for dietary management of AD. We hypothesize that the diet could inhibit AD-like pathologies in apoE4 mice, specifically cerebrovascular and connectivity impairment. Moreover, we evaluated the diet effect on cerebral blood flow (CBF, functional connectivity (FC, gray/white matter integrity, and postsynaptic density in aging apoE4 mice. At 10–12 months, apoE4 mice did not display prominent pathological differences compared to wild-type (WT mice. However, 16–18-month-old apoE4 mice revealed reduced CBF and accelerated synaptic loss. The diet increased cortical CBF and amount of synapses and improved white matter integrity and FC in both aging apoE4 and WT mice. We demonstrated that protective mechanisms on vascular and synapse health are enhanced by Fortasyn, independent of apoE genotype. We further showed the efficacy of a multimodal translational approach, including advanced MR neuroimaging, to study dietary intervention on brain structure and function in aging.

  5. A Dietary Treatment Improves Cerebral Blood Flow and Brain Connectivity in Aging apoE4 Mice.

    Wiesmann, Maximilian; Zerbi, Valerio; Jansen, Diane; Haast, Roy; Lütjohann, Dieter; Broersen, Laus M; Heerschap, Arend; Kiliaan, Amanda J


    APOE ε4 (apoE4) polymorphism is the main genetic determinant of sporadic Alzheimer's disease (AD). A dietary approach (Fortasyn) including docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium has been proposed for dietary management of AD. We hypothesize that the diet could inhibit AD-like pathologies in apoE4 mice, specifically cerebrovascular and connectivity impairment. Moreover, we evaluated the diet effect on cerebral blood flow (CBF), functional connectivity (FC), gray/white matter integrity, and postsynaptic density in aging apoE4 mice. At 10-12 months, apoE4 mice did not display prominent pathological differences compared to wild-type (WT) mice. However, 16-18-month-old apoE4 mice revealed reduced CBF and accelerated synaptic loss. The diet increased cortical CBF and amount of synapses and improved white matter integrity and FC in both aging apoE4 and WT mice. We demonstrated that protective mechanisms on vascular and synapse health are enhanced by Fortasyn, independent of apoE genotype. We further showed the efficacy of a multimodal translational approach, including advanced MR neuroimaging, to study dietary intervention on brain structure and function in aging.

  6. An approach to automatic blood vessel image registration of microcirculation for blood flow analysis on nude mice.

    Lin, Wen-Chen; Wu, Chih-Chieh; Zhang, Geoffrey; Wu, Tung-Hsin; Lin, Yang-Hsien; Huang, Tzung-Chi; Liu, Ren-Shyan; Lin, Kang-Ping


    Image registration is often a required and a time-consuming step in blood flow analysis of large microscopic video sequences in vivo. In order to obtain stable images for blood flow analysis, frame-to-frame image matching as a preprocessing step is a solution to the problem of movement during image acquisition. In this paper, microscopic system analysis without fluorescent labelling is performed to provide precise and continuous quantitative data of blood flow rate in individual microvessels of nude mice. The performance properties of several matching metrics are evaluated through simulated image registrations. An automatic image registration programme based on Powell's optimisation search method with low calculation redundancy was implemented. The matching method by variance of ratio is computationally efficient and improves the registration robustness and accuracy in practical application of microcirculation registration. The presented registration method shows acceptable results in close requisition to analyse red blood cell velocities, confirming the scientific potential of the system in blood flow analysis.

  7. Cathepsin K Deficiency Prevents the Aggravated Vascular Remodeling Response to Flow Cessation in ApoE-/- Mice

    Marjo M P C Donners; Bai, Lili; Lutgens, Suzanne P. M.; Wijnands, Erwin; Johnson, Jason; Schurgers, Leon J.; Liu, Cong-Lin; Daemen, Mat; Cleutjens, Kitty B.J.M.; Shi, Guo-Ping; BIESSEN, Erik; Heeneman, Sylvia


    Cathepsin K (catK) is a potent lysosomal cysteine protease involved in extracellular matrix (ECM) degradation and inflammatory remodeling responses. Here we have investigated the contribution of catK deficiency on carotid arterial remodeling in response to flow cessation in apoE-/- and wild type (wt) background. Ligation-induced hyperplasia is considerably aggravated in apoE-/- versus wt mice. CatK protein expression was significantly increased in neointimal lesions of apoE-/- compared with w...

  8. Spiny mice (Acomys cahirinus) do not respond to thymus-independent type 2 antigens.

    Pennello, Anthony; Taylor, Justin; Matlack, Robin; Karp, Jonathan; Riggs, James


    Analysis of the immune system of spiny mice (Acomys cahirinus) has been limited. Originally grouped with Mus, Acomys has recently been placed closer to Meriones (gerbils). This study compared immunity in Acomys, Mus, and Meriones. Lymphocytes from all rodents examined proliferated in response to mitogen and superantigen stimulation. Only Mus T cells responded to anti-CD3 stimulation. Acomys, like Meriones, and Mus that express xid, did not respond to thymus-independent type 2 antigens. Flow cytometric analyses revealed that T cell-specific MAbs did not bind Acomys or Meriones lymphocytes. The B cell-specific anti-CD45R (B220) MAb detected all rodent B cells and revealed the absence of a CD45R(lo) subset in the peritoneal cavity of Acomys and Meriones. Bone marrow from Acomys and Meriones failed to reconstitute B cell function in SCID mice. Thus, in terms of immunity, Acomys appears to be more similar to Meriones than Mus.

  9. Disturbed flow induces systemic changes in metabolites in mouse plasma: a metabolomics study using ApoE−/− mice with partial carotid ligation

    Go, Young-Mi; Kim, Chan Woo; Walker, Douglas I; Kang, Dong Won; Kumar, Sandeep; Orr, Michael; Uppal, Karan; Quyyumi, Arshed A.; Jo, Hanjoong; Jones, Dean P


    Disturbed blood flow (d-flow) occurring in branched and curved arteries promotes endothelial dysfunction and atherosclerosis, in part, by altering gene expression and epigenomic profiles in endothelial cells. While a systemic metabolic change is known to play a role in atherosclerosis, it is unclear whether it can be regulated by local d-flow. Here, we tested this hypothesis by carrying out a metabolomics study using blood plasma samples obtained from ApoE−/− mice that underwent a partial car...

  10. Cardiomyocyte regeneration from circulating bone marrow cells in mice.

    Kuramochi, Yukio; Fukazawa, Ryuji; Migita, Makoto; Hayakawa, Jun; Hayashida, Mari; Uchikoba, Yohko; Fukumi, Daichi; Shimada, Takashi; Ogawa, Shunichi


    We investigated the role of circulating bone marrow cells (BMC) in cardiomyocyte regeneration. BMC, isolated from transgenic mice expressing enhanced green fluorescent protein (GFP), were transplanted into lethally irradiated C57BL6 mice. Five weeks after bone marrow transplantation (BMT), flow cytometric analysis for GFP-positive cells confirmed reconstitution of transplanted bone marrow. Bone marrow transplant mice subsequently underwent left coronary artery ligation (myocardial infarction) or sham-operation, and were killed at 1 mo or 3 mo after operation. Infarct size was similar in bone marrow transplant mice at 1 mo (47.1 +/- 5.9%) and at 3 mo (45.3 +/- 7.8%), and echocardiography at 2 and 8 wk revealed decreasing left ventricular function. In infarcted heart, GFP-positive cells that expressed desmin and troponin T-C were identified by confocal microscopy. GFP and troponin T-C double-positive cells were predominantly in the peri-infarcted region (1 mo, 365 +/- 45 cells/50 sections; 3 mo: 458 +/- 100 cells/50 sections; p infarct, and sham-operated regions). Furthermore, BMC mobilization and differentiation into cardiomyocytes was found to be complete within 1 mo after myocardial infarction. These results demonstrate that circulating BMC undergo mobilization and differentiation in cardiac cells after myocardial infarction. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon.

  11. Immunotoxicity of the organochlorine pesticide methoxychlor in female ICR, BALB/c, and C3H/He mice.

    Hayashi, Koichi; Fukuyama, Tomoki; Ohnuma, Aya; Tajima, Yukari; Kashimoto, Yukiko; Yoshida, Toshinori; Kosaka, Tadashi


    Several types of pesticides, including organochlorines, are known to suppress or modulate immune responses. The present study evaluated the immunotoxicity of the organochlorine pesticide methoxychlor (MXC) in female BALB/c, C3H/He, and ICR mice. Mice were given oral MXC doses of 0, 30, 100, and 300 mg/kg each day for 7 consecutive days. On day 4, the mice also received an intravenous injection of sheep red blood cells (SRBC). The splenic plaque-forming cell (PFC) IgM response and the serum anti-SRBC IgM antibody titer were evaluated while splenic lymphocytes were counted by flow cytometry and the spleen underwent histopathological analysis. Significant decreases in IgM PFC responses were seen in BALB/c, C3H/He, and ICR mice that received MXC doses of 100 and 300 mg/kg. Similar changes in serum anti-SRBC IgM antibody titers occurred in three strain mice. Flow cytometric analysis revealed significantly decreased splenic T-cell (CD3+) populations in a dose dependent manner in BALB/c mice, and in the 300 mg/kg of MXC-treated group of C3H/He mice. Germinal center (GC) B-cell (CD19+PNA+) populations were significantly decreased in the 300 mg/kg of MXC-treated groups of all three mouse strains and in the 30 and 100 mg/kg of MXC-treated groups of BALB/c and C3H/He strain mice. Histopathological analysis revealed decreased cellularity of the periarteriolar lymphoid sheath (PALS; T-cell area) and decreased GC development in all three strains of mice treated with 300 mg/kg MXC. These results suggest that MXC has an immune-suppressive effect in mice, and that our protocol may be useful for rapidly detecting immunosuppression induced by environmental chemicals.

  12. Disturbed flow induces systemic changes in metabolites in mouse plasma: a metabolomics study using ApoE⁻/⁻ mice with partial carotid ligation.

    Go, Young-Mi; Kim, Chan Woo; Walker, Douglas I; Kang, Dong Won; Kumar, Sandeep; Orr, Michael; Uppal, Karan; Quyyumi, Arshed A; Jo, Hanjoong; Jones, Dean P


    Disturbed blood flow (d-flow) occurring in branched and curved arteries promotes endothelial dysfunction and atherosclerosis, in part, by altering gene expression and epigenomic profiles in endothelial cells. While a systemic metabolic change is known to play a role in atherosclerosis, it is unclear whether it can be regulated by local d-flow. Here, we tested this hypothesis by carrying out a metabolomics study using blood plasma samples obtained from ApoE(-/-) mice that underwent a partial carotid ligation surgery to induce d-flow. Mice receiving sham ligation were used as a control. To study early metabolic changes, samples collected from 1 wk after partial ligation when endothelial dysfunction occurs, but before atheroma develops, were analyzed by high-resolution mass spectrometry. A metabolome-wide association study showed that 128 metabolites were significantly altered in the ligated mice compared with the sham group. Of these, sphingomyelin (SM; m/z 703.5747), a common mammalian cell membrane sphingolipid, was most significantly increased in the ligated mice. Of the 128 discriminatory metabolites, 18 and 41 were positively and negatively correlated with SM, respectively. The amino acids methionine and phenylalanine were increased by d-flow, while phosphatidylcholine and phosphatidylethanolamine were decreased by d-flow, and these metabolites were correlated with SM. Other significantly affected metabolites included dietary and environmental agents. Pathway analysis showed that the metabolic changes of d-flow impacted broad functional networks. These results suggest that signaling from d-flow occurring in focal regions induces systemic metabolic changes associated with atherosclerosis.

  13. Decreased microvascular cerebral blood flow assessed by diffuse correlation spectroscopy after repetitive concussions in mice.

    Buckley, Erin M; Miller, Benjamin F; Golinski, Julianne M; Sadeghian, Homa; McAllister, Lauren M; Vangel, Mark; Ayata, Cenk; Meehan, William P; Franceschini, Maria Angela; Whalen, Michael J


    Repetitive concussions are associated with long-term cognitive dysfunction that can be attenuated by increasing the time intervals between concussions; however, biomarkers of the safest rest interval between injuries remain undefined. We hypothesize that deranged cerebral blood flow (CBF) is a candidate biomarker for vulnerability to repetitive concussions. Using a mouse model of human concussion, we examined the effect of single and repetitive concussions on cognition and on an index of CBF (CBFi) measured with diffuse correlation spectroscopy. After a single mild concussion, CBFi was reduced by 35±4% at 4 hours (Pconcussions spaced 1 day apart, CBFi was also reduced from preinjury levels 4 hours after each concussion but had returned to preinjury levels by 72 hours after the final concussion. Interestingly, in this repetitive concussion model, lower CBFi values measured both preinjury and 4 hours after the third concussion were associated with worse performance on the Morris water maze assessed 72 hours after the final concussion. We conclude that low CBFi measured either before or early on in the evolution of injury caused by repetitive concussions could be a useful predictor of cognitive outcome.

  14. Test-retest repeatability of myocardial blood flow and infarct size using {sup 11}C-acetate micro-PET imaging in mice

    Croteau, Etienne; Renaud, Jennifer M.; McDonald, Matthew; Klein, Ran; DaSilva, Jean N.; Beanlands, Rob S.B.; DeKemp, Robert A. [University of Ottawa Heart Institute, National Cardiac PET Centre, Ottawa, Ontario (Canada)


    Global and regional responses of absolute myocardial blood flow index (iMBF) are used as surrogate markers to assess response to therapies in coronary artery disease. In this study, we assessed the test-retest repeatability of iMBF imaging, and the accuracy of infarct sizing in mice using {sup 11}C-acetate PET. {sup 11}C-Acetate cardiac PET images were acquired in healthy controls, endothelial nitric oxide synthase (eNOS) knockout transgenic mice, and mice after myocardial infarction (MI) to estimate global and regional iMBF, and myocardial infarct size compared to {sup 18}F-FDG PET and ex-vivo histology results. Global test-retest iMBF values had good coefficients of repeatability (CR) in healthy mice, eNOS knockout mice and normally perfused regions in MI mice (CR = 1.6, 2.0 and 1.5 mL/min/g, respectively). Infarct size measured on {sup 11}C-acetate iMBF images was also repeatable (CR = 17 %) and showed a good correlation with the infarct sizes found on {sup 18}F-FDG PET and histopathology (r{sup 2} > 0.77; p < 0.05). {sup 11}C-Acetate micro-PET assessment of iMBF and infarct size is repeatable and suitable for serial investigation of coronary artery disease progression and therapy. (orig.)

  15. Laser speckle contrast imaging of blood flow from anesthetized mice: correcting drifts in measurements due to breathing movements

    Nogueira, Gesse E. C.; Ribeiro, Márcio A. C.; Campos, Juliane C.; Ferreira, Julio C. B.


    Background: Laser speckle contrast imaging allows non-invasive assessment of cutaneous blood flow. Although the technique is attractive to measure a quantity related to the skin blood flow (SBF) in anesthetized animal models, movements from breathing can mask the SBF signal. As a consequence, the measurement is overestimated because a variable amount of a DC component due to the breathing movements is added to the SBF signal. Objective: To evaluate a method for estimating the background level of the SBF signal, rejecting artefacts from breathing. Methods: A baseline correction method used for accurate DNA sequencing was evaluated, based on estimating the background level of a signal in small temporal sliding-windows. The method was applied to evaluate a mouse model of hindlimb ischemia. SBF signals from hindlimbs of anesthetized C57BL/6 mice (n=13) were registered. The mean SBF (Fi and Fc from ischemic and control hindlimbs) were computed from the registers and from the corresponding estimated background levels (Fib and Fcb from ischemic and control hindlimbs). Results: The mean values of the percentages (a measure of ischemia) MI = (Fi/Fc).100 and MIb = (Fib/Fcb).100 were computed to be 30+/-4% and 23+/-3% respectively (mean +/- SE). Evidences of statistical differences between both, ischemic and control hindlimbs, were obtained (p<0.05, paired student-t). The mean error [(MI-MIb)/MIb].100 obtained was 45+/-14% (mean+/-SE). Conclusion: The recovery of a corrupted SBF signal by breathing artefacts is feasible, allowing more accurate measurements.

  16. Contribuição da citometria de fluxo para o diagnóstico e prognóstico das síndromes mielodisplásicas The application of flow cytometric analysis of bone marrow cells for the diagnosis and prognosis of myelodysplastic syndromes

    Irene Lorand-Metze


    Full Text Available O diagnóstico das síndromes mielodisplásicas (SMD é baseado nos achados de citopenias no sangue periférico, na morfologia (atipias das células hemopoiéticas na medula óssea e no cariótipo. Em uma proporção considerável de casos, porém, o grau de atipias encontrado é discreto e sujeito a interpretações subjetivas. Além disso, alterações citogenéticas são encontradas apenas em 30%-80% dos casos. A citometria de fluxo multiparamétrica é uma técnica rápida, reproduzível e relativamente barata, capaz de objetivar alterações funcionais do clone SMD na maioria dos casos, o que permite o diagnóstico diferencial com patologias não-clonais que cursam com citopenias periféricas. Várias alterações têm sido descritas na expressão de antígenos ligados a linhagem e maturação celular nas três séries hemopoiéticas. Protocolos de três ou quatro cores analisando-se as séries eritroblástica, mielomonocítica e blastos têm sido propostos e conseguem resolver o diagnóstico diferencial em praticamente todos os casos. A citometria de fluxo também é útil para o acompanhamento dos pacientes, já que a progressão do clone neoplásico é acompanhada por um aumento do número de alterações fenotípicas e de células CD34+ além da diminuição de marcadores pró-apoptóticos.The diagnosis of MDS is based on the presence of peripheral cytopenias together with cell atypias in bone marrow precursors and cytogenetic abnormalities. However, in several cases, the cell atypias are discrete, and/or the karyotype is normal, precluding a clear-cut diagnosis. Multiparametric flow cytometry is a fast, reproducible and relatively inexpensive technique, which is able to disclose changes in the expression of lineage and maturation related antigens. Several of such abnormalities have been described in MDS. Three or four-color protocols have been used to analyze erythroblasts, granulocytes, monocytes and blasts, permitting, in most of the

  17. Cytometric analysis of shape and DNA content in mammalian sperm

    Gledhill, B.L.


    Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, of accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.

  18. C1galt1-deficient mice exhibit thrombocytopenia due to abnormal terminal differentiation of megakaryocytes.

    Kudo, Takashi; Sato, Takashi; Hagiwara, Kozue; Kozuma, Yukinori; Yamaguchi, Takashi; Ikehara, Yuzuru; Hamada, Michito; Matsumoto, Ken; Ema, Masatsugu; Murata, Soichiro; Ohkohchi, Nobuhiro; Narimatsu, Hisashi; Takahashi, Satoru


    C1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galt(flox) allele (Mx1-C1). Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIbα messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIbα protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of α- and β-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIbα in cells lacking O-glycan might be caused by increased proteolysis.

  19. Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice.

    Chie Amano

    Full Text Available To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice.

  20. Flow cytometric test using eosin-5′-maleimide (EMA) labelling of red blood for diagnosis of hereditary spherocytosis%流式细胞术检测伊红-5′-马来酰亚胺标记红细胞在80例遗传性球形红细胞增多症中的诊断价值

    王继英; 郑彬; 赵玉平; 陈雪晶; 刘燕; 薄丽津; 郑以州; 张凤奎; 汝昆


    目的 探讨伊红-5′-马来酰亚胺(EMA)为标记流式细胞术检测遗传性球形红细胞的灵敏性和特异性,并对试剂和样本稳定性进行验证.方法 对80例遗传性球形红细胞增多症(HS)和44例非HS患者外周血样本全部采用EMA标记流式细胞术检测、红细胞渗透脆性试验和酸化甘油溶解试验三种方法进行检测,比较三种方法的灵敏性和特异性,探讨EMA标记流式细胞术检测的可行性.并观察EMA及样本在不同储存条件下的稳定性.结果 通过检测124例样本得出,EMA标记流式细胞术检测的灵敏性和特异性分别为0.925和0.954,红细胞渗透脆性试验分别为0.950和0.455,酸化甘油溶解试验为1.000和0.318.红细胞渗透脆性试验和酸化甘油溶解试验的灵敏性稍高于EMA流式检测方法,但特异性差,不能明确区分球形红细胞增多是否为HS.EMA对温度很敏感,-80℃储存180 d较4℃存储1d稳定.HS样本的稳定性较好,4℃放置6d、室温条件放置3d不会影响结果的判定.结论 EMA标记流式细胞术检测HS具有良好的灵敏性和特异性,-80℃储存EMA较为稳定,须在样本4℃放置6d内、室温条件放置3d内完成检测.%Objective To investigate the sensitivity and specificity of eosin-5′-maleimide (EMA) assay for the diagnosis of hereditary spherocytosis (HS),and to verify the stability of reagent and samples.Methods EMA flow cytometry test,NaC1-osmotic fragility test and acidified glycerol lysis test were performed using peripheral blood samples from 80 patients with HS and 44 patients with other blood diseases,the sensitivity and specificity of the three methods were compared,and the feasibility of EMA binding test was estimated.The stability of EMA reagent and HS samples stored at different temperatures were tested.Results Among the 124 tested samples,the sensitivity and specificity of EMA binding test was 0.925 and 0.954,that of NaCl-osmotic fragility test was 0.950 and 0.455,and

  1. 多参数流式细胞术在多发性骨髓瘤及其微小残留疾病的免疫表型分析%Immunophenotypic analysis of multiparametric flow cytometric in multiple myeloma and minimal residual disease

    许艳丽; 王顺清; 毛平; 杜庆华


    Objective To investigate the detectable significance of multiparameter flow cytometry (MFC) for the first visiting and minimal residual disease (MRD) in the patients with multiple myeloma .Methods MFC was used to identify the plasma cells by the expression of CD138 or CD38 antigen in 74 patients with multiple myeloma .By combining surface antigens like CD45 ,CD56 , CD19 ,CD20 ,CD117 and the cytoplasm Kappa and Lambda light chain ,the aberrant myeloma cells were differentiated from normal plasma cells .Results In the 44 first visiting cases ,the positive expression of CD138 can be detected in all cases ,while the expres‐sion of CD19 was negative and 42 cases (95% ) were negative or weak positive expression for CD45 .The detection rates of CD38 , CD56 ,CD20 and CD117 were 98% ,93% ,45% and 41% ,respectively .The cytoplasm Kappa and Lambda light chains were showed the limited expression .Of the patients with MM ,14 cases were used for evaluating the change of immunophenotype at first visiting and during the treatment process ,among them ,11 cases(79% ) appeared the changes in at least one of aberrant phenotypes .4 cases (29% ) had the significant enhancement of antigen marker fluorescence intensities after chemotherapy and 7 cases (50% ) had sig‐nificant decrease of antigen marker fluorescence intensities after chemotherapy .CD45 ,CD19 and cytoplasm immunoglobulin light chains were the most stable marker ,no obvious antigen marker changes were found during the treatment ,while there was a signifi‐cant antigen density change in 2 cases of CD38 (14% ) ,7 cases of CD56 (50% ) ,4 cases of CD20 (29% ) and 2 cases of CD117 (14% ) .Of the 30 cases for evaluating MRD immunophenotype ,the abnormal myeloma cells were detected in 25 cases .In 5 cases ,no expression of limited Kappa and Lambda light chains was found and the ratio of Kappa and Lambda was 0 .5 - 2 ,which were identi‐fied as negative for MRD .Conclusion The multiparameter flow cytometry has important

  2. Environmental cold exposure increases blood flow and affects pain sensitivity in the knee joints of CFA-induced arthritic mice in a TRPA1-dependent manner.

    Fernandes, Elizabeth S; Russell, Fiona A; Alawi, Khadija M; Sand, Claire; Liang, Lihuan; Salamon, Robin; Bodkin, Jennifer V; Aubdool, Aisah A; Arno, Matthew; Gentry, Clive; Smillie, Sarah-Jane; Bevan, Stuart; Keeble, Julie E; Malcangio, Marzia; Brain, Susan D


    The effect of cold temperature on arthritis symptoms is unclear. The aim of this study was to investigate how environmental cold affects pain and blood flow in mono-arthritic mice, and examine a role for transient receptor potential ankyrin 1 (TRPA1), a ligand-gated cation channel that can act as a cold sensor. Mono-arthritis was induced by unilateral intra-articular injection of complete Freund's adjuvant (CFA) in CD1 mice, and in mice either lacking TRPA1 (TRPA1 KO) or respective wildtypes (WT). Two weeks later, nociception and joint blood flow were measured following exposure to 10 °C (1 h) or room temperature (RT). Primary mechanical hyperalgesia in the knee was measured by pressure application apparatus; secondary mechanical hyperalgesia by automated von Frey system; thermal hyperalgesia by Hargreaves technique, and weight bearing by the incapacitance test. Joint blood flow was recorded by full-field laser perfusion imager (FLPI) and using clearance of (99m)Technetium. Blood flow was assessed after pretreatment with antagonists of either TRPA1 (HC-030031), substance P neurokinin 1 (NK1) receptors (SR140333) or calcitonin gene-related peptide (CGRP) (CGRP8-37). TRPA1, TAC-1 and CGRP mRNA levels were examined in dorsal root ganglia, synovial membrane and patellar cartilage samples. Cold exposure caused bilateral primary mechanical hyperalgesia 2 weeks after CFA injection, in a TRPA1-dependent manner. In animals maintained at RT, clearance techniques and FLPI showed that CFA-treated joints exhibited lower blood flow than saline-treated joints. In cold-exposed animals, this reduction in blood flow disappears, and increased blood flow in the CFA-treated joint is observed using FLPI. Cold-induced increased blood flow in CFA-treated joints was blocked by HC-030031 and not observed in TRPA1 KOs. Cold exposure increased TRPA1 mRNA levels in patellar cartilage, whilst reducing it in synovial membranes from CFA-treated joints. We provide evidence that environmental

  3. Simplified flow cytometric assay to detect minimal residual disease in childhood with acute lymphoblastic leukemia Detecção de doença residual mínima em crianças com leucemia linfoblástica aguda por citometria de fluxo

    Elizabete Delbuono


    Full Text Available The detection of minimal residual disease (MRD is an important prognostic factor in childhood acute lymphoblastic leukemia (ALL providing crucial information on the response to treatment and risk of relapse. However, the high cost of these techniques restricts their use in countries with limited resources. Thus, we prospectively studied the use of flow cytometry (FC with a simplified 3-color assay and a limited antibody panel to detect MRD in the bone marrow (BM and peripheral blood (PB of children with ALL. BM and PB samples from 40 children with ALL were analyzed on days (d 14 and 28 during induction and in weeks 24-30 of maintenance therapy. Detectable MRD was defined as > 0.01% cells expressing the aberrant immunophenotype as characterized at diagnosis among total events in the sample. A total of 87% of the patients had an aberrant immunophenotype at diagnosis. On d14, 56% of the BM and 43% of the PB samples had detectable MRD. On d28, this decreased to 45% and 31%, respectively. The percentage of cells with the aberrant phenotype was similar in both BM and PB in T-ALL but about 10 times higher in the BM of patients with B-cell-precursor ALL. Moreover, MRD was detected in the BM of patients in complete morphological remission (44% on d14 and 39% on d28. MRD was not significantly associated to gender, age, initial white blood cell count or cell lineage. This FC assay is feasible, affordable and readily applicable to detect MRD in centers with limited resources.A detecção de doença residual mínima (DRM é um importante fator prognóstico na leucemia linfóide aguda (LLA infantil e fornece informações sobre a resposta ao tratamento e o risco de recaída. Entretanto, os altos custos das técnicas utilizadas limitam seu uso nos países em desenvolvimento. Desta forma, realizamos um estudo prospectivo para avaliar a citometria de fluxo (CF, utilizando três fluorescências e um painel limitado de anticorpos monoclonais, como método de detec

  4. Safrole-modulated immune response is mediated through enhancing the CD11b surface marker and stimulating the phagocytosis by macrophages in BALB/c mice.

    Fan, M-J; Lin, S-Y; Yu, C-C; Tang, N-Y; Ho, H-C; Chung, H-K; Yang, J-S; Huang, Y-P; Ip, S-W; Chung, J-G


    Safrole, a component of piper betle inflorescence, is a documented rodent hepatocarcinogen and inhibits bactericidal activity and the release of superoxide anion (O(2-)) by polymorphonuclear leukocytes (PMNs). In the present study, we investigated the effects of safrole on immune responses, including natural killer (NK) cell cytotoxicity, phagocytic activity and population distribution of leukocytes from normal BALB/c mice. The cells population (cell surface markers) and phagocytosis by macrophages and monocytes from the peripheral blood mononuclear cells (PBMCs) were determined, and NK cell cytotoxicity from splenocytes of mice after oral treatment with safrole was performed using flow cytometric assay. Results indicated that safrole did not affect the weights of body, spleen and liver when compared with the normal mice group. Safrole also promoted the levels of CD11b (monocytes) and Mac-3 (macrophages) that might be the reason for promoting the activity of phagocytosis. However, safrole reduced the cell population such as CD3 (T cells) and CD19 (B cells) of safrole-treated normal mice by oral administration. Furthermore, safrole elevated the uptake of Escherichia coli-labelled fluorescein isothiocyanate (FITC) by macrophages from blood and significantly stimulated the NK cell cytotoxicity in normal mice in vivo. In conclusions, alterations of the cell population (the increase in monocytes and macrophages, respectively) in safrole-treated normal BALB/c mice might indirectly influence the immune responses in vivo.

  5. Donor MHC gene to mitigate rejection of transplantation in recipient mice

    LI Tong; ZHANG Zhi-tai; LI Hui; YAN Jun; TAN Jia-li; L(U) Yue-ping; HOU Sheng-cai; LI Shen-tao; XU Qing; TONG Xue-hong; DING Jie


    Background Donor organ rejection continues to be a significant problem for patients receiving transplants.We therefore tested whether transferring a donor's major histocompatibility complex (MHC) gene to the recipient would mitigate the rejection of transplanted hearts in mice.Methods H-2Kkgene from donor mice was amplified using nested polymerase chain reaction (PCR) and ligated into a mammalian expression vector,which was then transfected into thymus ground mass cells collected from the recipients.Clones stably expressing the transgene were then injected into the recipients' thymus visualized using ultrasound.Control mice were administered cells previously transfected with empty vector.Following heart transplantation,cardiac activity was monitored electrocardiographically.Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests.Finally,the transplanted hearts were sectioned,stained and examined under light microscopy.Results Southern analysis following nested PCR revealed clear expression of H-2Kk gene.Following transplantation,electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2Kk than in those receiving control cells.Flow cytometric analysis using an anti-H-2Kk antibody confirmed its expression in H-2Kk treated recipients but not in control mice.Mixed lymphocyte cultures containing H-2Kk treated cells showed significantly less proliferation than those containing control cells.Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2Kk treated mice and large areas of necrosis.Conclusion Rejection of transplanted hearts can be mitigated substantially by introducing the donor's MHC into the recipient.

  6. Flow karyotyping and sorting of human chromosomes

    Gray, J.W.; Lucas, J.; Peters, D.; Pinkel, D.; Trask, B.; van den Engh, G.; Van Dilla, M.A.


    Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purification of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.

  7. Bone marrow deficiency of MCPIP1 results in severe multi-organ inflammation but diminishes atherogenesis in hyperlipidemic mice.

    Fang Yu

    Full Text Available OBJECTIVE: MCPIP1 is a newly identified protein that profoundly impacts immunity and inflammation. We aim to test if MCPIP1 deficiency in hematopoietic cells results in systemic inflammation and accelerates atherogenesis in mice. APPROACH AND RESULTS: After lethally irradiated, LDLR(-/- mice were transplanted with bone marrow cells from either wild-type or MCPIP1(-/- mice. These chimeric mice were fed a western-type diet for 7 weeks. We found that bone marrow MCPIP1(-/- mice displayed a phenotype similar to that of whole body MCPIP1(-/- mice, with severe systemic and multi-organ inflammation. However, MCPIP1(-/- bone marrow recipients developed >10-fold less atherosclerotic lesions in the proximal aorta than WT bone marrow recipients, and essentially no lesions in en face aorta. The diminishment in atherosclerosis in bone marrow MCPIP1(-/- mice may be partially attributed to the slight decrease in their plasma lipids. Flow cytometric analysis of splenocytes showed that bone marrow MCPIP1(-/- mice contained reduced numbers of T cells and B cells, but increased numbers of regulatory T cells, Th17 cells, CD11b+/Gr1+ cells and CD11b+/Ly6C(low cells. This overall anti-atherogenic leukocyte profile may also contribute to the reduced atherogenesis. We also examined the cholesterol efflux capability of MCPIP1 deficient macrophages, and found that MCPIP1 deficiency increased cholesterol efflux to apoAI and HDL, due to increased protein levels of ABCA1 and ABCG1. CONCLUSIONS: Hematopoietic deficiency of MCPIP1 resulted in severe systemic and multi-organ inflammation but paradoxically diminished atherogenesis in mice. The reduced atheroegensis may be explained by the decreased plasma cholesterol levels, the anti-atherogenic leukocyte profile, as well as enhanced cholesterol efflux capability. This study suggests that, while atherosclerosis is a chronic inflammatory disease, the mechanisms underlying atherogenesis-associated inflammation in arterial wall

  8. A dominated and resistant subpopulation causes regrowth after response to 1,3-bis(2-chloroethyl)-1-nitrosourea treatment of a heterogeneous small cell lung cancer xenograft in nude mice

    Aabo, K; Roed, H; Vindeløv, L L


    ) and a resistant (NYH) tumor were used to produce mixed solid tumors in nude mice. Mixtures of 592/NYH (9:1 and 1:1) were inoculated s.c. After 3-4 weeks of tumor growth, the mice were stratified according to tumor size and randomized to treatment with BCNU 40 mg/kg i.p. (10% of lethal dose) or no treatment. Tumor...... growth curves were used to calculate the effect of the treatment, and changes in the relative proportions of 592 and NYH in the mixed tumors were monitored by flow cytometric DNA analysis by which the two cell lines were distinguishable due to differences in DNA content. A significant response...... was eradicated. These results indicate that resistant and undetectable (dominated) subpopulations in heterogeneous tumors may be responsible for relapse and that the fractional size and the growth characteristics of the resistant subpopulation may determine the magnitude of the clinical response to cytotoxic...

  9. Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes

    Cremers, Eline M.P.; Westers, Theresia M.; Alhan, Canan; Cali, Claudia; Visser-Wisselaar, Heleen A.; Chitu, Dana A.; van der Velden, Vincent H.J.; te Marvelde, Jeroen G.; Klein, Saskia K.; Muus, Petra; Vellenga, Edo; de Greef, Georgina E.; Legdeur, Marie-Cecile C.J.C.; Wijermans, Pierre W.; Stevens-Kroef, Marian J.P.L.; da Silva-Coelho, Pedro; Jansen, Joop H.; Ossenkoppele, Gert J.; van de Loosdrecht, Arjan A.


    Flow cytometric analysis is a recommended tool in the diagnosis of myelodysplastic syndromes. Current flow cytometric approaches evaluate the (im)mature myelo-/monocytic lineage with a median sensitivity and specificity of ~71% and ~93%, respectively. We hypothesized that the addition of erythroid lineage analysis could increase the sensitivity of flow cytometry. Hereto, we validated the analysis of erythroid lineage parameters recommended by the International/European LeukemiaNet Working Group for Flow Cytometry in Myelodysplastic Syndromes, and incorporated this evaluation in currently applied flow cytometric models. One hundred and sixty-seven bone marrow aspirates were analyzed; 106 patients with myelodysplastic syndromes, and 61 cytopenic controls. There was a strong correlation between presence of erythroid aberrancies assessed by flow cytometry and the diagnosis of myelodysplastic syndromes when validating the previously described erythroid evaluation. Furthermore, addition of erythroid aberrancies to two different flow cytometric models led to an increased sensitivity in detecting myelodysplastic syndromes: from 74% to 86% for the addition to the diagnostic score designed by Ogata and colleagues, and from 69% to 80% for the addition to the integrated flow cytometric score for myelodysplastic syndromes, designed by our group. In both models the specificity was unaffected. The high sensitivity and specificity of flow cytometry in the detection of myelodysplastic syndromes illustrates the important value of flow cytometry in a standardized diagnostic approach. The trial is registered at as NTR1825; EudraCT n.: 2008-002195-10 PMID:27658438

  10. Label-free in vivo optical micro-angiography imaging of cerebral capillary blood flow within meninges and cortex in mice with the skull left intact

    Jia, Yali; Wang, Ruikang K.


    Abnormal microcirculation within meninges is common in many neurological diseases. There is a need for an imaging method that is capable of visualizing functional meningeal microcirculations alone, preferably decoupled from the cortical blood flow. Optical microangiography (OMAG) is a recently developed label-free imaging method capable of producing 3D images of dynamic blood perfusion within micro-circulatory tissue beds at an imaging depth up to ~2 mm, with an unprecedented imaging sensitivity to the blood flow at ~4 μm/s. In this study, we demonstrate the utility of ultra-high sensitive OMAG in imaging the detailed blood flow distributions, at a capillary level resolution, within meninges and cortex in mice with the cranium left intact. The results indicate that OMAG can be a valuable tool for the study of meningeal circulations.

  11. Flow cytometric detection of some activation and proliferation markers in human hematopoietic cell lines.

    Glasová, M; Koníková, E; Kusenda, J; Babusíková, O


    Simultaneous surface marker/DNA, cytoplasmic/DNA or nuclear/DNA staining was used to study proliferation of hematopoietic cell lines (MOLT4, BJAB, P3HR1). Different fixation/permeabilization methods (paraformaldehyde with metanol or Tween 20 or saponin, buffered formaldehyde-acetone) were used providing optimal results of the double stainings. There was a significant increase of S phase and proliferation index (PI) of CD71+ and Ki67+ MOLT4 cells in comparison with their negative counterparts. This indicates their close connection with proliferation. Unlike that, the correlation between the expression of CD38 and S phase or PI was not significant either in MOLT4 or in P3HRI cells. For cytoplasmic markers CD3 (in MOLT4 cells) and CD22 (in BJAB cells) statistically significant (cCD3) and not significant (cCD22) correlation was demonstrated between their expression and S phase or PI. Molecular equivalents of soluble fluorescein values for CD71 were always higher than for CD38. The density of these cell surface markers in addition to the percentage of their expression is of considerable significance for their evaluation as activation or proliferation markers.

  12. Flow Cytometric Analysis of DNA Content in Parotid Tumor and Its Contiguous Acini

    ZHU Shengrong; SHAO Lenan; CHEN Weimin; WU Huihua; WANG Xiuli; CHEN Xinming


    To investigate the relationship between proliferative capacity of salivary gland cells in contiguous acini of parotid tumors and recurrent neoplasma, DNA contents of 30 fresh specimens of parotid were studied by using cytometry in tumors, normal and shallow or deep lobe acini of the masses. The results showed that the DI was 1.369, S % 16.95, PI 26.18 in malignant tumors;DI was 1.171, S % 12.41, PI 15.54 in recurrent pleomorphic adenoma; DI was 1.141, S % 12.74, PI 13.07 in pleomorphic adenoma, DI was 0.999, S % 5.10, PI 8.00 in normal acini. Analysis of variance showed there was a significant difference (P<0.01). The average DNA contents of shallow on deep lobe of contiguous tumors was 1.08 in DI, 10. 65 in S %, 13.49 in PI in malignant tumor, 1.06 in DI, 8.96 in S % and 9.85 in PI in pleomorphic adenoma, which were all higher than in normal acini (P>0.05). It was concluded that the levels of DI and S % of parotid tumor and its contiguous acini are related to degree of malignancy or recurrent condition of the tumors, suggesting contiguous acini of parotid tumors had the strong capacity of proliferation,which might play an important role in recurrent or malignant change of the parotid tumors.

  13. Joint modeling and registration of cell populations in cohorts of high-dimensional flow cytometric data.

    Pyne, Saumyadipta; Lee, Sharon X; Wang, Kui; Irish, Jonathan; Tamayo, Pablo; Nazaire, Marc-Danie; Duong, Tarn; Ng, Shu-Kay; Hafler, David; Levy, Ronald; Nolan, Garry P; Mesirov, Jill; McLachlan, Geoffrey J


    In biomedical applications, an experimenter encounters different potential sources of variation in data such as individual samples, multiple experimental conditions, and multivariate responses of a panel of markers such as from a signaling network. In multiparametric cytometry, which is often used for analyzing patient samples, such issues are critical. While computational methods can identify cell populations in individual samples, without the ability to automatically match them across samples, it is difficult to compare and characterize the populations in typical experiments, such as those responding to various stimulations or distinctive of particular patients or time-points, especially when there are many samples. Joint Clustering and Matching (JCM) is a multi-level framework for simultaneous modeling and registration of populations across a cohort. JCM models every population with a robust multivariate probability distribution. Simultaneously, JCM fits a random-effects model to construct an overall batch template--used for registering populations across samples, and classifying new samples. By tackling systems-level variation, JCM supports practical biomedical applications involving large cohorts. Software for fitting the JCM models have been implemented in an R package EMMIX-JCM, available from

  14. Generation, culture and flow-cytometric characterization of primary mouse macrophages.

    Schleicher, Ulrike; Bogdan, Christian


    Macrophages are not only host cells for many pathogens, but also fulfill several key functions in the innate and adaptive immune response, including the release of pro- and anti-inflammatory cytokines, the generation of organic and inorganic autacoids, the phagocytosis and killing of intracellular microorganisms or tumor cells, and the degradation and presentation of antigens. Several of these functions are shared by other immune cells, including dendritic cells, granulocytes, NK cells, and/or T lymphocytes. Thus, the analysis of macrophage functions in vitro using primary mouse cell populations requires standardized methods for the generation and culture of macrophages that guarantee high cell purity as well as the absence of stimulatory microbial contaminants. This chapter presents methodology to achieve these aims.

  15. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

    Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P


    Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce...

  16. Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni

    We developed a method to measure ingestion and digestion rates of bacterivorous protists feeding on pathogenic bacteria. We tested this method using the enteric bacteria Campylobacter jejuni and a freshwater colpodid ciliate. Campylobacter and a non-pathogenic bacteria isolated from the environment ...

  17. Measurement of Transient Permeability of Sp2/0 Myeloma Cells: Flow Cytometric Study

    Novickij Vitalij


    Full Text Available Electroporation is an electric field induced phenomenon occurring when the permeability of the cell membrane is increased due to the excess of critical transmembrane potential. Fluorescent dye assays are frequently used for evaluation of the permeabilization rate, however, the protocols vary, which negatively affects the repeatability of the results. In this work we have designed experiments to investigate the protocols and threshold concentrations of the Propidium Iodide (PI and YO-PRO-1 (YP fluorescent dyes for evaluation of mammalian cell permeabilization induced by electroporation. The Sp2/0 mouse myeloma cells were used and the bursts of 100 μs × 8 electrical pulses of 0.8-2 kV/cm were applied. It has been shown that the dye concentration has an influence on the detectable permeabilization, and the concentrations below 30 μM for PI and 1 μM for YP should be avoided for measurement of electropermeabilization efficacy due to unreliable fluorescence signals. Further, based on the experimental data, the permeabilization curve for the Sp2/0 myeloma cells in the 0.8-2 kV/cm range has been presented.

  18. Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

    De Keersmaecker, Sigrid C. J.; Braeken, Kristien; Verhoeven, Tine L. A.; Perea Vélez, Mónica; Lebeer, Sarah; Vanderleyden, Jos; Hols, Pascal


    Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 104 transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus ...

  19. Preanalytical requirements for flow cytometric evaluation of platelet activation: choice of anticoagulant.

    Mody, M; Lazarus, A H; Semple, J W; Freedman, J


    Accurate assessment of in vivo or in vitro platelet activation requires optimal preanalytical conditions to prevent artefactual in vitro activation of the platelets. The choice of anticoagulant is one of the critical preanalytical conditions as anticoagulants exert different effects on the activation of platelets ex vivo. We tested the effectiveness of Diatube-H (also known as CTAD; sodium citrate, theophylline, adenosine and dipyridamole) and citrate vacutainer tubes in preventing artefactual activation of platelets and preserving functional reserve. Platelet surface expression of the CD62P (reflecting alpha granule release), CD63 (reflecting lysosomal release) and modulation of normal platelet membrane glycoproteins CD41a and CD42b, were measured in whole blood and in isolated platelets immediately after collection and at 6, 24 and 48 h after venipuncture. Samples taken into Diatube-H showed less spontaneous platelet activation than did those taken into citrate. To measure in vitro platelet functional reserve, thrombin was added as agonist to blood stored for varying periods up to 48 h. Although Diatube-H suppressed in vitro platelet activation for up to 4 h, in samples kept for 6-24 h before thrombin addition, the inhibitory effect was lost and platelets responded fully to agonist activation. Hence, Diatube-H preserved platelets and allowed for measurement of in vivo platelet activation as well as thrombin-induced in vitro platelet activation after 6-24 h, in both whole blood and isolated platelets.

  20. Flow cytometric analysis of RNA synthesis by detection of bromouridine incorporation

    Larsen, J K; Jensen, Peter Østrup; Larsen, J


    RNA synthesis has traditionally been investigated by a laborious and time-consuming radiographic method involving incorporation of tritiated uridine. Now a faster non-radioactive alternative has emerged, based on immunocytochemical detection. This method utilizes the brominated RNA precursor...... bromouridine, which is taken into a cell, phosphorylated, and incorporated into nascent RNA. The BrU-substituted RNA is detected by permeabilizing the cells and staining with certain anti-BrdU antibodies. This dynamic approach yields information complementing that provided by cellular RNA content analysis...

  1. Flow cytometric investigation of immune-response-related surface molecules on human colorectal cancers

    Diederichsen, Axel Cosmus Pyndt; Stenholm, A C; Kronborg, O;


    Our purpose was to clarify whether human colorectal cancer cells are equipped to present tumour-associated-antigens to the immune system, and whether this ability correlates with lymphoid infiltration, the Dukes' stage and Jass classification. Enzymatically dissociated tumour cells from 70...... molecules, but not the class II, was correlated with lymphoid infiltration and the Jass classification. Expression of these surface molecules was not correlated with the Dukes' stage. The tumour cells were generally equipped to present antigens to the effector arm of the immune system since HLA class I...... is expressed, but the tumour cells were not optimal in stimulating an immune response, since HLA class II and CD58 were only marginally expressed and CD80 and CD54 were absent....

  2. Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis.

    Wieser, Andreas; Storz, Enno; Liegl, Gabriele; Peter, Annabell; Pritsch, Michael; Shock, Jonathan; Wai, Sun Nyunt; Schubert, Sören


    There currently exists no efficient and easy method for size profiling and counting of membranous nano-scale particles, such as bacterial outer membrane vesicles (OMVs). We present here a cost-effective and fast method capable of profiling and counting small sample volumes of nano-scale membranous vesicles with standard laboratory equipment without the need for any washing steps. OMV populations of different bacterial species are compared and even subpopulations of OMVs can be identified after a simple labelling procedure. Counting is possible over three orders of magnitude without any changes to the protocol. Protein contaminations do not alter the described measurements. Copyright © 2014 Elsevier GmbH. All rights reserved.

  3. The effect of light level, CO2 flow rate, and anesthesia on the stress response of mice during CO2 euthanasia.

    Powell, Karin; Ethun, Kelly; Taylor, Douglas K


    Euthanasia protocols are designed to mitigate the stress experienced by animals, and an environment that induces minimal stress helps achieve that goal. A protocol that is efficient and practical in a typical animal research facility is also important. Light intensity, isoflurane, and CO2 flow rate were studied for their impact on the stress response of mice during CO2 euthanasia. Behavior was observed and scored during euthanasia and serum corticosterone was measured immediately after death. Unsurprisingly, animals euthanized with a high-flow rate of CO2 became unconscious in the least amount of time, while animals euthanized with a low-flow rate required the most time to reach unconsciousness. There was a significant increase in anxious behaviors in animals in the isoflurane group (F1,12 = 6.67, P = 0.024), the high-flow rate CO2 group (F1,12 = 10.24, P = 0.007), and bright chamber group (F1,12 = 7.27, P = 0.019). Serum corticosterone was highest in the isoflurane group (124.72 ± 83.98 ng/ml), however there was no significant difference in corticosterone levels observed for the other study variables of light and flow-rate. A darkened chamber and low CO2 flow rates help to decrease stress experienced during CO2 euthanasia, while the use of isoflurane was observed to increase the stress response during euthanasia.

  4. Effects of 60 Hz electromagnetic field exposure on testicular germ cell apoptosis in mice

    JinSangLee; SangSeokAhn; KyeongCheonJung; Yoon-WonKim; SangKonLee


    Aim:To evaluate the effects of 60 Hz extremely low frequency (ELF) elelctromagnetic field (EMF) exposure on germ cell apoptosis in the testis of mice.Methods:Adult male BALB/c mice (7 weeks of age) were exposed to a 60 Hz EMF of 0.1 mT or 0.5 mT for 24 h/day.A sham-exposed group served as the control.After 8 weeks of exposure,the mice were sacrificed.Germ cell apoptosis in the testis was assessed by histopathological examination,the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) and flow cytometric examination of isolated spermatogenic cells stained with 7 aminoactinomycin D (7-AAD).Results:EMF exposure did not significantly affect the body and testis weights,but significantly increased the incidence of germ cell death.The distinguishing morphological feature of EMF exposure was a decrement in the number of well organized seminiferous tubules.Quantitative analysis of TUNEL-positive germ cells showed a significantly higher apoptotic rate in the 0.5 mT exposed mice than that in the sham controls (P<0.05),while the difference between the two exposed groups was insignificant.The TUNEL-positive cells were mainly spermatogonia.In flow cytometryanalysis,the percentage of live cells [forward scatter count (FSC)hgh7-AAD-] was lower in the exposed groups than that in the controls (Figure 5A),but the decrease in viability was not statistically significant.Conclusion:Continuousexposure to ELF EMF may induce testicular germ cell apoptosis in mice.(Asian J Androl 2004 Mar;6:29-34)

  5. Automation in high-content flow cytometry screening.

    Naumann, U; Wand, M P


    High-content flow cytometric screening (FC-HCS) is a 21st Century technology that combines robotic fluid handling, flow cytometric instrumentation, and bioinformatics software, so that relatively large numbers of flow cytometric samples can be processed and analysed in a short period of time. We revisit a recent application of FC-HCS to the problem of cellular signature definition for acute graft-versus-host-disease. Our focus is on automation of the data processing steps using recent advances in statistical methodology. We demonstrate that effective results, on par with those obtained via manual processing, can be achieved using our automatic techniques. Such automation of FC-HCS has the potential to drastically improve diagnosis and biomarker identification.

  6. Cytometric analysis of DNA changes induced by sulfur mustard

    Smith, W.J.; Sanders, K.M.; Ruddle, S.E.; Gross, C.L.


    Sulfur mustard is an alkylating agent which causes severe, potentially debilitating blisters following cutaneous exposure. Its mechanism of pathogenesis is unknown and no antidote exists to prevent its pathology. The biochemical basis of sulfur mustard's vesicating activity has been hypothesized to be a cascade of events beginning with alkylation of DNA. Using human cells in culture, we have assessed the effects of sulfur mustard on cell cycle activity using flow cytometry with propidium iodide. Two distinct patterns emerged, a Gl/S interface block at concentrations equivalent to vesicating doses (>50-micronM) and a G2 block at 10-fold lower concentrations. In addition, noticeable increases in amount of dye uptake were observed at 4 and 24 hours after sulfur mustard exposure. These increases are believed to be related to DNA repair activities and can be prevented by treatment of the cells with niacinamide, which inhibits DNA repair. Other drugs which provide alternate alkylating sites or inhibit cell cycle progression were shown to lower the cytotoxicity of sulfur mustard and to protect against its direct DNA damaging effects.

  7. Effects of undenatured whey protein supplementation on CXCL12- and CCL21-mediated B and T cell chemotaxis in diabetic mice

    Badr Gamal


    Full Text Available Abstract Background Long and persistent uncontrolled diabetes tends to degenerate the immune system and leads to an increased incidence of infection. Whey proteins (WPs enhance immunity during early life and have a protective role in some immune disorders. In this study, the effects of camel WP on the chemotaxis of B and T cells to CXCL12 and CCL21 in diabetic mice were investigated. Results Flow cytometric analysis of the surface expressions of CXCR4 (CXCL12 receptor and CCR7 (CCL21 receptor on B and T cells revealed that the surface expressions of CXCR4 and CCR7 were not significantly altered in diabetic and WP-supplemented diabetic mice compared with control mice. Nevertheless, B and T lymphocytes from diabetic mice were found to be in a stunned state, with a marked and significant (P Conclusion Our data revealed the benefits of WP supplementation in enhancing cytoskeletal rearrangement and chemotaxis in B and T cells, and subsequently improving the immune response in diabetic mice.

  8. Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice.

    Lin, Kou-Ray; Yang-Yen, Hsin-Fang; Lien, Huang-Wei; Liao, Wei-Hao; Huang, Chang-Jen; Lin, Liang-In; Li, Chung-Leung; Yen, Jeffrey Jong-Young


    Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.

  9. [The specific features of diagnosis of mixed-phenotype acute leukemia: A combination of B-cell antigen expressions according to the results of flow cytometry and morphological markers of myeloid differentiation in blast cells: A clinical case].

    Gritsaev, S V; Kostroma, I I; Ryadnova, G M; Tiranova, S A; Chubukina, Zh V; Balashova, V A; Zenina, M N; Martynkevich, I S; Potikhonova, N A; Abdulkadyrov, K M


    This rare type of acute leukemia, blast cells of which express myeloid and/or lymphoid markers, is mainly diagnosed using flow cytometric findings. The paper describes a clinical case of mixed-phenotype acute leukemia, in which B-cell lymphoid antigen expressions were revealed by a flow cytometric technique, while bone marrow morphological specimens showed the signs of myeloid differentiation specific to blast cells. It is concluded that there is a need for a comprehensive examination of patients with new-onset acute leukemia and for an aggregate analysis of flow cytometric results with morphological and cytochemical findings.

  10. Accelerated wound healing phenotype in Interleukin 12/23 deficient mice

    Matias Marie AT


    Full Text Available Abstract Background The concept that a strong inflammatory response involving the full complement of cytokines and other mediators is critical for unimpaired healing has been challenged by wound healing studies using transgenic and knockout (KO mice. The present study explored the effect of abrogation of the p40 subunit, which is shared by the pro-inflammatory cytokines interleukin (IL-12 and IL-23, on wound closure of excisional oral mucosal wounds. Methods Double IL-12 and IL-23 KO mice and C57BL ⁄ 6J wildtype mice were wounded on the dorsal surface of the tongue using a 2 mm biopsy punch. The degree of epithelialization was examined histologically. At specific timepoints wounds were examined for cellular and molecular markers for inflammation and angiogenesis using 1 immunohistochemistry; 2 analysis of RNA expression; and 3 flow cytometric analysis. Results Compared to wild type controls, KO mice displayed enhanced healing, which was driven by a greater influx of neutrophils and macrophages during the early stages of wound healing, and increased induction of messenger RNA (mRNA for endothelial derived neutrophil attractant (ENA78 chemokine and macrophage inflammatory protein-2 alpha (MIP-2α. Increased mRNA for monocyte-attracting chemokines including monocyte chemoattractant protein (MCP-1 and MCP-3 was seen from day 1, together with higher levels of IL-1β and IL-6 within 24 hours after wounding. In addition, mRNA for vascular endothelial growth factor (VEGF-A was upregulated in KO mice within 2 hours after injury, and higher expression of this mediator was confirmed by immunohistochemistry. Conclusion Overall, the accelerated oral mucosal wound healing seen in IL-12/IL-23p40 KO compared to wildtype mice was associated with the early establishment of an inflammatory response and vascularization.

  11. Potential Use of Quantum Dots in Flow Cytometry

    Raquel Ibáñez-Peral


    Full Text Available QDs may offer significant advantages in environmental and bead-based applications where the target cells need to be discriminated above background fluorescence. We have examined the possible applications of QDs for flow cytometric measurements (FCM by studying their excitation - emission spectra and their binding to paramagnetic beads. We labelled beads with either QDs or a commonly-used fluorochrome (FITC and studied their fluorescence intensity by FCM. Flow cytometric comparisons indicated that the minimum fluorophore concentration required for detection of QDs above autofluorescent background was 100-fold less than for FITC.

  12. Elimination of young erythrocytes from blood circulation and altered erythropoietic patterns during paraquat induced anemic phase in mice.

    Bhardwaj, Nitin; Saxena, Rajiv K


    Paraquat a widely used herbicide causes a variety of toxic effects on humans and animals. The present study is focused on the interaction of paraquat with the mouse erythroid system. Administration of paraquat (10 mg/kg body weight i.p. on alternate days in C57Bl/6 mice) induced a significant fall in blood erythrocyte count on 7, 14, and 21 day time points but the erythrocyte count reverted back to normal by 28th day indicating the emergence of refractoriness to paraquat. A marked surge in the blood reticulocyte count was observed in paraquat treated mice that also subsided by 28th day. Young erythrocytes in circulation were randomly eliminated from blood circulation in paraquat treated mice and a significant elevation in the level of reactive oxygen species (ROS) was also observed maximally the erythrocytes of this age group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were identified and enumerated flow cytometrically based on their expression of Ter119 and transferrin (CD71) receptor. Proliferative activity of erythroid cells, their relative proportion as well as their absolute numbers fell significantly in bone marrow of paraquat treated mice but all these parameters were significantly elevated in spleens of paraquat treated mice. These changes were essentially restricted to the cells belonging to the two earliest stages of erythroid differentiation. Taken together our results indicate that paraquat treatment causes a transient anemia in mice resulting from random elimination of young circulating erythrocytes as well as depressed erythropoietic activity in bone marrow. Spleen erythropoietic activity however was elevated in paraquat treated mice.

  13. Preparation of myeloid derived suppressor cells (MDSC) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (AutoMACS).

    Nelson, Nadine; Szekeres, Karoly; Cooper, Denise; Ghansah, Tomar


    sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1(+) leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1(+) enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.

  14. Role of capsule and suilysin in mucosal infection of complement-deficient mice with Streptococcus suis.

    Seitz, Maren; Beineke, Andreas; Singpiel, Alena; Willenborg, Jörg; Dutow, Pavel; Goethe, Ralph; Valentin-Weigand, Peter; Klos, Andreas; Baums, Christoph G


    Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3(-/-) mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3(-/-) mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3(-/-) mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3(-/-) blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR(-/-) mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

  15. Valsartan ameliorates podocyte loss in diabetic mice through the Notch pathway.

    Gao, Feng; Yao, Min; Cao, Yanping; Liu, Shuxia; Liu, Qingjuan; Duan, Huijun


    The Notch pathway is known to be linked to diabetic nephropathy (DN); however, its underlying mechanism was poorly understood. In the present study, we examined the effect of Valsartan, an angiotensin II type 1 receptor antagonist, on the Notch pathway and podocyte loss in DN. Diabetes was induced in mice by an intraperitoneal injection of streptozotocin and and this was followed by treatment with Valsartan. Levels of blood glucose, kidney weight and body weight, as well as proteinuria were measured. Samples of the kidneys were also histologically examined. The relative levels of Jagged1, Notch1, Notch intracellular domain 1 (NICD1), Hes family BHLH transcription factor 1 (Hes1) and Hes-related family BHLH transcription factor with YRPW motif 1 expression (Hey1) in the glomeruli were determined by immunohistochemical analysis, western blot analysis and RT-qPCR. The B-Cell CLL/Lymphoma 2 (Bcl-2) and p53 pathways were examined by western blot analysis. Apoptosis and detachment of podocytes from the glomerular basement membrane were examined using a TUNEL assay, flow cytometric analysis and ELISA. The number of podocytes was quantified by measuring Wilms tumor-1 (WT-1) staining. We noted that the expression of Jagged1, Notch1, NICD1, Hes1 and Hey1 was increased in a time-dependent manner in the glomeruli of mice with streptozotocin (STZ)-induced diabetes. Moreover, in diabetic mice, Valsartan significantly reduced kidney weight and proteinuria, and mitigated the pathogenic processes in the kidneys. Valsartan also inhibited the activation of Notch, Bcl-2 and p53 pathways and ameliorated podocyte loss in the glomeruli of mice with STZ-induced diabetes. Taken together, these findings indicated that Valsartan exerted a beneficial effect on reducing podocyte loss, which is associated with inhibition of Notch pathway activation in the glomeruli of diabetic mice.

  16. Curcumin improves learning and memory ability and its neuroprotective mechanism in mice

    PAN Rui; QIU Sheng; LU Da-xiang; DONG Jun


    Background Increasing evidence suggests that many neurons may die through apoptosis in Alzheimer's disease (AD).Mitochondrial dysfunction has been implicated in this process of neuronal cell death. One promising approach for preventing AD is based upon anti-apoptosis to decrease death of nerve cells. In this study, we observed the memory improving properties of curcumin in mice and investigated the neuroprotective effect of curcumin in vitro and in vivo.Methods The mice were given AlCl3 orally and injections of D-galactose intraperitoneally for 90 days to establish the AD animal model. From day 45, the curcumin group was treated with curcumin for 45 days. Subsequently, the step-through test, neuropathological changes in the hippocampus and the expression of Bax and Bcl-2 were carried out to evaluate the effect of curcumin on the AD model mice. In cultured PC12 cells, AlCl3 exposure induced apoptosis. The MTT assay was used to measure cell viabilities; flow cytometric analysis to survey the rate of cell apoptosis; DNA-binding fluorochrome Hoechst 33258 to observe nuclei changes in apoptotic cells and Western blot analysis of Bax, Bcl-2 to investigate the mechanisms by which curcumin protects cells from toxicity.Results Curcumin significantly improved the memory ability of AD mice in the step-through test, as indicated by the reduced number of step-through errors (P 0.05). AlCl3 significantly reduced the viability of PC12 cells (P 0.05).Conclusions This study demonstrates that curcumin improves the memory ability of AD mice and inhibits apoptosis in cultured PC12 cells induced by AlCl3. Its mechanism may involve enhancing the level of Bcl-2.

  17. Cytometric analysis of surface molecules of leucocytes and phagocytic activity of granulocytes and monocytes/macrophages in cows with pyometra.

    Brodzki, P; Kostro, K; Brodzki, A; Niemczuk, K; Lisiecka, U


    Pyometra is a serious problem in dairy cow herds, causing large economic losses due to infertility. The development of pyometra depends mainly on the immunological status of the cow. The aim of the study was a comparative evaluation of selected indicators involving non-specific and specific immunity in cows with pyometra and in cows without inflammation of the uterus. The study was performed in 20 cows, which were divided into two groups: pyometra group and healthy group, each comprising 10 cows, based on the results of cytological and ultrasonographic tests. A flow cytometric analysis was performed for the surface molecules CD4, CD8, CD14, CD21, CD25 and CD4(+) CD25(+) on leucocytes, and the phagocytic activity was determined from granulocytes and monocytes/macrophages in the peripheral blood and uterine washings, respectively. It was demonstrated that the percentage of phagocytic granulocytes and monocytes/macrophages in both the peripheral blood and uterine washings was significantly lower in cows with pyometra compared with the healthy group (p < 0.001). Significantly (p ≤ 0.001) lower percentage of CD4(+) , CD14(+) , CD25(+) and CD4(+) CD25(+) phenotype leucocytes was also observed in the peripheral blood of cows from the pyometra group, along with a significantly higher (p < 0.001) percentage of CD8(+) and CD21(+) lymphocytes as compared to the healthy group. The results of work indicate that disfunction of cell immunity coexisting with pyometra may be caused by a bacterial infection and the presence of blocking agents (IL-10), released by the increasing number of CD8(+) lymphocytes what leads to the advanced inflammation of uterus.

  18. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    Chesney, Alden; Good, David; Reis, Marciano


    Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

  19. In vivo immunomodulatory effects of the methanolic leaf extract of Gymnema sylvestre in Swiss albino mice

    Ahirwal Laxmi


    Full Text Available In the present study we performed a comparative phytochemical analysis of the immunomodulating activities of the methanol leaf extract of Gymnema sylvestre (MLEGS in Swiss albino mice. The phytochemical screening conducted on MLEGS revealed the presence of several phytoconstituents, including saponins, alkaloids, glycosides, phenols, tannins, and flavonoids. Immunomodulatory activities were determined by hemagglutination antibody (HA titer and delayed-type hypersensitivity (DTH tests for determining specific and non-specific immune responses. Flow cytometric techniques were performed for the estimation of B lymphocytes (CD3 and CD19 and Th2 cytokines (IL-2, IFN-γ and IL-4. The response produced by oral administration of MLEGS elicited a significant reduction in a dose-related manner in the primary and secondary antibody response and DTH response. The response produced by oral administration of MLEGS elicited significant reduction in a dose-related manner in the primary and secondary antibody and DTH responses, with maximum reduction observed at 200 mg/kg-body wt. The maximal reductions in the production of CD3, CD19, IL-2, IFN-γ and IL-4 were 31.59, 32.12, 29.51, 32.45 and 33.53%, respectively, at 200 mg/kg body weight. This study demonstrates that G. sylvestre exerts immunosuppressive effects on the components of the immune system of mice, and points to its significant immunomodulatory potential.

  20. Fisetin-Rich Extracts of Rhus verniciflua Stokes Improve Blood Flow Rates in Mice Fed Both Normal and High-Fat Diets.

    Im, Won Kyun; Park, Hyun Jung; Lee, Kwang Soo; Lee, Jung Hoon; Kim, Young Dong; Kim, Kyeong-Hee; Park, Sang-Jae; Hong, Seokmann; Jeon, Sung Ho


    Although it has been previously reported that Rhus verniciflua Stokes (RVS) possesses in vitro anti-inflammatory activity, the precise in vivo mechanisms of RVS extracts and a main active component called fisetin have not been well elucidated. In this study, using newly developed protocols, we prepared urushiol-free but fisetin-enriched RVS extracts and investigated their effects on the vascular immune system. We found that the water-soluble fractions of detoxified RVS with the flavonoid fisetin can inhibit lipopolysaccharide-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). Furthermore, RVS can reduce inducible nitric oxide synthase and COX2 gene expression levels, which are responsible for NO and PGE2 production, respectively, in RAW264.7 macrophage cells. Because inflammation is linked to the activation of the coagulation system, we hypothesized that RVS and its active component fisetin possess anticoagulatory activities. As expected, we found that both RVS and fisetin could inhibit the coagulation of human peripheral blood cells. Moreover, in vivo RVS treatment could return the retarded blood flow elicited by a high-fat diet (HFD) back to the normal level in mice. In addition, RVS treatment has significantly reduced body weight gained by HFD in mice. Taken together, the fisetin-rich RVS extracts have potential antiplatelet and antiobesity activities and could be used as a functional food ingredient to improve blood circulation.

  1. Electroacupuncture acutely improves cerebral blood flow and attenuates moderate ischemic injury via an endothelial mechanism in mice.

    Ji Hyun Kim

    Full Text Available Electroacupuncture (EA is a novel therapy based on traditional acupuncture combined with modern eletrotherapy that is currently being investigated as a treatment for acute ischemic stroke. Here, we studied whether acute EA stimulation improves tissue and functional outcome following experimentally induced cerebral ischemia in mice. We hypothesized that endothelial nitric oxide synthase (eNOS-mediated perfusion augmentation was related to the beneficial effects of EA by interventions in acute ischemic injury. EA stimulation at Baihui (GV20 and Dazhui (GV14 increased cerebral perfusion in the cerebral cortex, which was suppressed in eNOS KO, but there was no mean arterial blood pressure (MABP response. The increased perfusion elicited by EA were completely abolished by a muscarinic acetylcholine receptor (mAChR blocker (atropine, but not a β-adrenergic receptor blocker (propranolol, an α-adrenergic receptor blocker (phentolamine, or a nicotinic acetylcholine receptor (nAChR blocker (mecamylamine. In addition, EA increased acetylcholine (ACh release and mAChR M3 expression in the cerebral cortex. Acute EA stimulation after occlusion significantly reduced infarct volume by 34.5% when compared to a control group of mice at 24 h after 60 min-middle cerebral artery occlusion (MCAO (moderate ischemic injury, but not 90-min MCAO (severe ischemic injury. Furthermore, the impact of EA on moderate ischemic injury was totally abolished in eNOS KO. Consistent with a smaller infarct size, acute EA stimulation led to prominent improvement of neurological function and vestibule-motor function. Our results suggest that acute EA stimulation after moderate focal cerebral ischemia, but not severe ischemia improves tissue and functional recovery and ACh/eNOS-mediated perfusion augmentation might be related to these beneficial effects of EA by interventions in acute ischemic injury.

  2. CD8{sup +}CD25{sup +} T cells reduce atherosclerosis in apoE(−/−) mice

    Zhou, Jianchang; Dimayuga, Paul C.; Zhao, Xiaoning; Yano, Juliana; Lio, Wai Man; Trinidad, Portia; Honjo, Tomoyuki; Cercek, Bojan; Shah, Prediman K.; Chyu, Kuang-Yuh, E-mail:


    Highlights: •The role of a sub-population of CD8{sup +} T cells with suppressor functions was investigated in atherosclerosis. •CD8{sup +}CD25{sup +} T cells from adult apoE(−/−) mice had phenotype characteristics of T suppressor cells. •These CD8{sup +}CD25{sup +} T cells reduced CD4{sup +} T cell proliferation and CD8{sup +} cytotoxic activity in vitro. •Adoptive transfer of CD8{sup +}CD25{sup +} T cells significantly reduced atherosclerosis. •CD8{sup +}CD25{sup +} T cells have a suppressive function in atherosclerosis. -- Abstract: Background: It is increasingly evident that CD8{sup +} T cells are involved in atherosclerosis but the specific subtypes have yet to be defined. CD8{sup +}CD25{sup +} T cells exert suppressive effects on immune signaling and modulate experimental autoimmune disorders but their role in atherosclerosis remains to be determined. The phenotype and functional role of CD8{sup +}CD25{sup +} T cells in experimental atherosclerosis were investigated in this study. Methods and results: CD8{sup +}CD25{sup +} T cells were observed in atherosclerotic plaques of apoE(−/−) mice fed hypercholesterolemic diet. Characterization by flow cytometric analysis and functional evaluation using a CFSE-based proliferation assays revealed a suppressive phenotype and function of splenic CD8{sup +}CD25{sup +} T cells from apoE(−/−) mice. Depletion of CD8{sup +}CD25{sup +} from total CD8{sup +} T cells rendered higher cytolytic activity of the remaining CD8{sup +}CD25{sup −} T cells. Adoptive transfer of CD8{sup +}CD25{sup +} T cells into apoE(−/−) mice suppressed the proliferation of splenic CD4{sup +} T cells and significantly reduced atherosclerosis in recipient mice. Conclusions: Our study has identified an athero-protective role for CD8{sup +}CD25{sup +} T cells in experimental atherosclerosis.

  3. Deciphering the neuronal circuitry controlling local blood flow in the cerebral cortex with optogenetics in PV::Cre transgenic mice

    Alan eUrban


    Full Text Available Although it is know since more than a century that neuronal activity is coupled to blood supply regulation, the underlying pathways remains to be identified. In the brain, neuronal activation triggers a local increase of cerebral blood flow (CBF that is controlled by the neurogliovascular unit composed of terminals of neurons, astrocytes and blood vessel muscles. It is generally accepted that the regulation of the neurogliovascular unit is adjusted to local metabolic demand by local circuits. Today experimental data led us to realize that the regulatory mechanisms are more complex and that a neuronal system within the brain is devoted to the control of local brain blood flow. Recent optogenetic experiments combined with functional magnetic resonance imaging (fMRI have revealed that light stimulation of neurons expressing the calcium binding protein parvalbumin (PV is associated with positive blood oxygen level-dependent (BOLD signal in the corresponding barrel field but also with negative BOLD in the surrounding deeper area. Here, we demonstrate that in acute brain slices, ChR2-based photostimulation of PV containing neurons gives rise to an effective contraction of penetrating arterioles. These results support the neurogenic hypothesis of a complex distributed nervous system controlling the CBF.

  4. Assessment of Equine Autoimmune Thrombocytopenia (EAT by flow cytometry

    Schwarzwald Colin


    Full Text Available Abstract Rationale Thrombocytopenia is a platelet associated process that occurs in human and animals as result of i decreased production; ii increased utilization; iii increased destruction coupled to the presence of antibodies, within a process know as immune-mediated thrombocytopenia (IMT; or iv platelet sequestration. Thus, the differentiation of the origin of IMT and the development of reliable diagnostic approaches and methodologies are important in the clarification of IMT pathogenesis. Therefore, there is a growing need in the field for easy to perform assays for assessing platelet morphological characteristics paired with detection of platelet-bound IgG. Objectives This study is aimed to develop and characterize a single color flow cytometric assay for detection of platelet-bound IgG in horses, in combination with flow cytometric assessment of platelet morphological characteristics. Findings The FSC and SSC evaluation of the platelets obtained from the thrombocytopenic animals shows several distinctive features in comparison to the flow cytometric profile of platelets from healthy animals. The thrombocytopenic animals displayed i increased number of platelets with high FSC and high SSC, ii a significant number of those gigantic platelets had strong fluorescent signal (IgG bound, iii very small platelets or platelet derived microparticles were found significantly enhanced in one of the thrombocytopenic horses, iv significant numbers of these microplatelet/microparticles/platelet-fragments still carry very high fluorescence. Conclusions This study describes the development and characterization of an easy to perform, inexpensive, and noninvasive single color flow cytometric assay for detection of platelet-bound IgG, in combination with flow cytometric assessment of platelet morphological characteristics in horses.

  5. DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in Ptch(flox/flox)CD4Cre(+/-) mice.

    Uhmann, Anja; Heß, Ina; Frommhold, Anke; König, Simone; Zabel, Sebastian; Nitzki, Frauke; Dittmann, Kai; Lühder, Fred; Christiansen, Hans; Reifenberger, Julia; Schulz-Schaeffer, Walter; Hahn, Heidi


    The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

  6. [Inhibition Function of Dominant-negative Mutant Gene Survivin-D53A to SPC-A1 Lung Adenocarcinoma Xenograft in Nude Mice Models].

    Yu, Min; Peng, Xingchen; Lu, You; Huang, Meijuan


    Survivin-D53A (SVV-D53A) is a dominant-negative mutant survivin, which represents a potential promising target for cancer gene therapy. The present study was designed to determine whether SVV-D53A plasmid encapsuled by DOTAP: Chol liposome would have the anti-tumor activity against SPC-A1 lung adenocarcinoma, and to detect the possible mechanisms. In our experiment, SPC-A1 cells were transfected in vitro with SVV-D53A plasmid and examined for protein expression by Western blot, then flow cytometric analysis was used to detect apoptosis. SPC-A1 lung adenocarcinoma xenografts were established in vivo in the nude mice, which received the i. v. administrations of SVV-D53A plasmid/liposome complexes. After mice were sacrificed, the paraffin-embedded tumor tissue sections were used for proliferating cell nuclear antigen (PCNA) expression and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Compared with the control group, the mice treated with SVV-D53A plasmid had an obviously reduced tumor volume, with high level of apoptosis and decreased cell proliferation in tumor tissue. The research results proved that the administration of SVV-D53A plasmid resulted in significant inhibition of SPC-A1 cells both in vitro and in vivo. The functional mechanism is that the anti-tumor response causes and induces tumor cell apoptosis.

  7. Characterization of the T61 human breast carcinoma established in nude mice

    Brünner, N; Bastert, G B; Poulsen, H S


    and flow cytometric DNA analysis to be aneuploid. By electron microscopy, the tumour cells were shown to display the characteristics of glandular epithelium; a light microscopic examination revealed morphological characteristics similar to those of an axillary metastasis of the patient from whom the T61...

  8. Failure of effector function of human CD8+ T Cells in NOD/SCID/JAK3⁻/⁻ immunodeficient mice transplanted with human CD34+ hematopoietic stem cells.

    Yoshinori Sato

    Full Text Available Humanized mice, which are generated by transplanting human CD34+ hematopoietic stem cells into immunodeficient mice, are expected to be useful for the research on human immune responses. It is reported that antigen-specific T cell responses occur in immunodeficient mice transplanted with both human fetal thymus/liver tissues and CD34+ fetal cells, but it remains unclear whether antigen-specific T cell responses occur in those transplanted with only human CD34+ hematopoietic stem cells (HSCs. Here we investigated the differentiation and function of human CD8+ T cells reconstituted in NOD/SCID/Jak3⁻/⁻ mice transplanted with human CD34+ HSCs (hNOK mice. Multicolor flow cytometric analysis demonstrated that human CD8+ T cells generated from the CD34+ HSCs comprised only 3 subtypes, i.e., CD27(highCD28+CD45RA+CCR7+, CD27+CD28+CD45RA⁻CCR7+, and CD27+CD28+CD45RA⁻CCR7⁻and had 3 phenotypes for 3 lytic molecules, i.e., perforin(Per⁻granzymeA(GraA⁻granzymeB(GraB⁻, Per⁻GraA+GraB⁻, and Per(lowGraA+GraB+. These CD8+ T cells failed to produce IFN-γ and to proliferate after stimulation with alloantigens. These results indicate that the antigen-specific T cell response cannot be elicited in mice transplanted with only human CD34+ HSCs, because the T cells fail to develop normally in such mice.

  9. Improved graft survival in highly sensitized patients undergoing renal transplantation after the introduction of a clinically validated flow cytometry crossmatch.

    Limaye, Sandhya


    Flow cytometric techniques are increasingly used in pretransplant crossmatching, although there remains debate regarding the clinical significance and predictive value of donor-specific antibodies detected by flow cytometry. At least some of the discrepancies between published studies may arise from differences in cutoffs used and lack of standardization of the test.

  10. Coenzyme Q10 suppresses Th17 cells and osteoclast differentiation and ameliorates experimental autoimmune arthritis mice.

    Jhun, JooYeon; Lee, Seung Hoon; Byun, Jae-Kyeong; Jeong, Jeong-Hee; Kim, Eun-Kyung; Lee, Jennifer; Jung, Young-Ok; Shin, Dongyun; Park, Sung Hwan; Cho, Mi-La


    Coenzyme Q10 (CoQ10) is a lipid-soluble antioxidant synthesized in human body. This enzyme promotes immune system function and can be used as a dietary supplement. Rheumatoid arthritis (RA) is an autoimmune disease leading to chronic joint inflammation. RA results in severe destruction of cartilage and disability. This study aimed to investigate the effect of CoQ10 on inflammation and Th17 cell proliferation on an experimental rheumatoid arthritis (RA) mice model. CoQ10 or cotton seed oil as control was orally administrated once a day for seven weeks to mice with zymosan-induced arthritis (ZIA). Histological analysis of the joints was conducted using immunohistochemistry. Germinal center (GC) B cells, Th17 cells and Treg cells of the spleen tissue were examined by confocal microscopy staining. mRNA expression was measured by real-time PCR and protein levels were estimated by enzyme-linked immunosorbent assay (ELISA). Flow cytometric analysis (FACS) was used to evaluate Th17 cells and Treg cells. CoQ10 mitigated the severity of ZIA and decreased serum immunoglobulin concentrations. CoQ10 also reduced RANKL-induced osteoclastogenesis, inflammatory mediators and oxidant factors. Th17/Treg axis was reciprocally controlled by CoQ10 treatment. Moreover, CoQ10 treatment on normal mouse and human cells cultured in Th17 conditions decreased the number of Th17 cells and enhanced the number of Treg cells. CoQ10 alleviates arthritis in mice with ZIA declining inflammation, Th17 cells and osteoclast differentiation. These findings suggest that CoQ10 can be a potential therapeutic substance for RA.

  11. Lymphocyte activation and hepatic cellular infiltration in immunocompetent mice infected by dengue virus.

    Chen, Hsuen-Chin; Lai, Show-Yun; Sung, Jui-Min; Lee, Shu-Hwae; Lin, Yu-Chin; Wang, Wei-Kung; Chen, Yee-Chun; Kao, Chuan-Liang; King, Chwan-Chuen; Wu-Hsieh, Betty A


    Activation and expansion of dengue virus-specific T cells and abnormal liver functions in dengue patients have been documented. However, it remains to be determined whether T cells are involved in the pathogenic mechanism of dengue virus infection. In this study, immunocompetent C57BL/6 mice were employed to study dengue virus-induced T cell activation. Mice were inoculated with 10(8) PFU dengue virus serotype 2 strain 16681 by the intravenous route. Dengue viral core RNA was detected by RT-PCR in mouse serum, liver, spleen, and brain at different time points after infection. Splenic T cells were activated as evidenced by their expression of CD69 and O-glycosylated CD43 at as early as day 3 after infection. Splenic T cell expression of O-glycosylated CD43 and IFN-gamma production coordinately peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed the infiltrating CD8(+) T cell to CD4(+) T cell ratio was 5/3. After a second inoculation of dengue virus, hepatic T cell infiltration and liver enzyme levels increased sharply. The infiltrating hepatic CD8(+) T cell to CD4(+) T cell ratio increased to 5.8/1. A strong correlation was found between T cell activation and hepatic cellular infiltration in immunocompetent mice infected with dengue virus. The kinetics of liver enzyme elevation also correlated with that of T cell activation. These data suggest a relationship between T cell infiltration and elevation of liver enzymes.

  12. Expression of Cyclins A, E and Topoisomerase II α correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination

    Laytragoon-Lewin Nongnit


    Full Text Available Abstract Background The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role. Results Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II α showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells. Conclusions Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA

  13. IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis.

    Mémet, S; Laouini, D; Epinat, J C; Whiteside, S T; Goudeau, B; Philpott, D; Kayal, S; Sansonetti, P J; Berche, P; Kanellopoulos, J; Israël, A


    Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.

  14. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  15. CD4+FoxP3+ regulatory T cells from Gαi2-/- mice are functionally active in vitro, but do not prevent colitis.

    Yu-Yuan C Götlind

    Full Text Available BACKGROUND: Mice deficient in the inhibitory G protein subunit Gαi2 spontaneously develop a T helper 1 dominated colitis. We examined whether a defect in CD4(+FoxP3(+ regulatory T cells (Treg underpins the pathogenesis of colitis in the Gαi2(-/- (Gαi2-deficient colitis model. METHODOLOGY/PRINCIPAL FINDINGS: Using flow cytometry, we found that thymus and colonic lamina propria, but not spleen and mesenteric lymph nodes, of colitic Gαi2(-/- mice contained increased frequencies of Treg, whereas FoxP3 expression intensity was similar in Gαi2(-/- compared to Gαi2(+/- or Gαi2(+/+ wild type (WT mice. The frequency of CD4(+FoxP3(+ T cells expressing CD103 was significantly increased in Gαi2(-/- compared to WT mice. Treg in colons from WT mice clustered in the T cell areas of colonic lymphoid patches (CLP, with relatively few Treg in the lamina propria, as demonstrated by immunohistochemistry. In Gαi2(-/- mice, CLP were not observed but lamina propria Treg were increased in number and frequency within the CD4(+ infiltrate, compared to WT mice. Using an in vitro co-culture system and flow cytometric analysis of cell division we could demonstrate that the in vitro suppressive function of WT and Gαi2(-/- CD4(+FoxP3(+ regulatory T cells (WT-Treg and KO-Treg was indistinguishable, but that T effector cells (CD4(+25(- T cells from Gαi2(-/- mice were less readily suppressed than WT effectors (WT-Teff by Treg from either source. However, neither WT nor Gαi2(-/- Treg was able to suppress colitis induced by adoptive transfer of Gαi2(-/- effector T cells (KO-Teff to RAG2(-/- recipients. The enhanced inflammatory activity of Gαi2(-/- effectors was accompanied by increased expression of an effector/memory T cell phenotype and increased cytokine secretion, especially IL-4, IL-6 and IFN-γ. CONCLUSIONS: There is an increased frequency of Gαi2(-/- Treg in the colon, and they demonstrate no endogenous functional defect. However, Gαi2(-/- T effector cells

  16. Continuous flow microcalorimetric measurement of heat production in white adipose tissue from obese (ob/ob) mice and their lean littermates.

    Hansen, E S; Knudsen, J


    Heat production, free fatty acid and glycerol release from white adipose tissue fat pads from obese (ob/ob) mice and their lean littermates are determined. Heat production was significantly lower in obese mice compared to lean mice when expressed on wet weight basis but not when expressed on DNA basis. Noradrenaline significantly increased the heat production in fat pads from both groups of animals. However, the increase in heat production due to noradrenaline addition in fat pads from lean mice was significantly higher than in fat pads from obese mice. The release of free fatty acids and glycerol before incubation with noradrenaline was similar from fat pads from both groups of animals. Addition of noradrenaline to the fat pads increased the release of free fatty acids and glycerol in both groups of animals, but the increase was significantly larger from fat pads from lean mice. In the absence of noradrenaline the free fatty acid/glycerol ratio (mol/mol) in the effluent was 7.9:1 and 4.8:1 for lean mice and obese mice, respectively. In the presence of noradrenaline the ratio decreased to 3:1 for both groups of animals.

  17. Miniaturized flow cytometer with 3D hydrodynamic particle focusing and integrated optical elements applying silicon photodiodes

    Rosenauer, M.; Buchegger, W.; Finoulst, I.; Verhaert, P.D.E.M.; Vellekoop, M.


    In this study, the design, realization and measurement results of a novel optofluidic system capable of performing absorbance-based flow cytometric analysis is presented. This miniaturized laboratory platform, fabricated using SU-8 on a silicon substrate, comprises integrated polymer-based waveguide

  18. Genome-size variation in switchgrass (Panicum virgatum): flow cytometry and cytology reveal rampant aneuploidy

    Switchgrass (Panicum virgatum L.), a native perennial dominant of the prairies of North America, has been targeted as a model herbaceous species for biofeedstock development. A flow-cytometric survey of a core set of 11 primarily upland polyploid switchgrass accessions indicated that there was con...

  19. Impaired Mobilization of Vascular Reparative Bone Marrow Cells in Streptozotocin-Induced Diabetes but not in Leptin Receptor-Deficient db/db Mice.

    Vasam, Goutham; Joshi, Shrinidh; Jarajapu, Yagna P R


    Diabetes is associated with impaired mobilization of bone marrow stem/progenitor cells that accelerate vascularization of ischemic areas. This study characterized mobilization of vascular reparative bone marrow progenitor cells in mouse models of diabetes. Age-matched control or streptozotocin (STZ)-induced diabetic, and db/db mice with lean-controls were studied. Mobilization induced by G-CSF, AMD3100 or ischemia was evaluated by flow cytometric enumeration of circulating Lin(-)Sca-1(+)cKit(+) (LSK) cells, and by colony forming unit (CFU) assay. The circulating WBCs and LSKs, and CFUs were reduced in both models with a shorter duration (10-12 weeks) of diabetes compared to their respective controls. Longer duration of STZ-diabetes (≥20 weeks) induced impairment of G-CSF- or AMD3100-mobilization (P mobilization by G-CSF or AMD3100 was either increased or unaffected (P mobilization, of LSK cells were impaired in both models. Leptin receptor antagonist, PESLAN-1, increased G-CSF- or AMD3100-mobilization of WBCs and LSKs, compared to the untreated. Leptin increased basal WBCs, decreased basal and AMD3100-mobilized LSK cells, and had no effect on G-CSF. These results suggest that mobilopathy is apparent in STZ-diabetes but not in db/db mice. Leptin receptor antagonism would be a promising approach for reversing diabetic bone marrow mobilopathy.

  20. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    Bienenmann-Ploum, M.E.; Huet, A.C.; Campbell, K.; Fodey, T.L.; Vincent, U.; Delahaut, P.; Elliot, C.T.; Nielen, M.W.F.


    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive

  1. Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion.

    Rustagi, Arjun; Doehle, Brian P; McElrath, M Juliana; Gale, Michael


    Interferon regulatory factor-3 (IRF-3) is a master transcription factor that drives the host intracellular innate immune response to virus infection. The importance of IRF-3 in innate immune responses is highlighted by the fact that pathogenic viruses have developed strategies for antagonism of IRF-3. Several tools exist for evaluation of viral regulation of IRF-3 activation and function, but high-quality monoclonal antibodies that mark the differential activation states of human IRF-3 are lacking. To study IRF-3 activation, turnover, and depletion in a high-throughput manner in the context of virus infection, we have developed two new monoclonal antibodies to human IRF-3. These antibodies detect IRF-3 in virus-infected cells in a wide variety of assays and provide a new tool to study virus-host interactions and innate immune signaling.

  2. Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies.

    Araghi, Atefeh; Nassiri, Seyed Mahdi; Atyabi, Nahid; Rahbarghazi, Reza; Mohammadi, Elham


    There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45.

  3. Selection of fluorescent probes for flow cytometric viability assessment of Listeria monocytogenes exposed to membrane-active and oxidizing disinfectants

    Luppens, S.B.I.; Barbaras, B.; Breeuwer, P.; Rombouts, F.M.; Abee, T.


    The aim of this study was to select fluorescence methods for use as alternatives to plate counting to assess the viability of Listeria monocytogenes cells exposed to benzalkonium chloride (BAC) and hydrogen peroxide, two disinfectants with different mechanisms of action. A further aim of this study

  4. Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions.

    del Olmo, David; Parrilla, Inmaculada; Sanchez-Osorio, Jonatan; Gomis, Jesus; Angel, Miguel A; Tarantini, Tatiana; Gil, Maria A; Cuello, Cristina; Vazquez, Jose L; Roca, Jordi; Vaquez, Juan M; Martinez, Emilio A


    The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10(6) unsorted sperm per oviduct; (2) single LI with 0.5 × 10(6) sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10(6) sex-sorted sperm per oviduct and 0.5 × 10(6) sex-sorted sperm per uterine horn. The farrowing rates were lower (P sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10(6) sperm per oviduct and 1 × 10(6) sperm per uterine horn; or (2) 1 × 10(6) sperm per oviduct and 2 × 10(6) sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10(6) sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.

  5. Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.

    del Olmo, D; Parrilla, I; Gil, M A; Maside, C; Tarantini, T; Angel, M A; Roca, J; Martinez, E A; Vazquez, J M


    The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies.

  6. Flow cytometric sexing of spider sperm reveals an equal sperm production ratio in a female-biased species

    Vanthournout, Bram; Deswarte, K; Hammad, H


    -determining sperm cells; thus bias in sperm production does not contribute to the sex ratio bias observed in this species. This demonstrates that other factors such as parental genes suppressing endosymbiont effects and cryptic female choice might play a role in sex allocation in this species.......Producing equal amounts of male and female offspring has long been considered an evolutionarily stable strategy. Nevertheless, exceptions to this general rule (i.e. male and female biases) are documented in many taxa, making sex allocation an important domain in current evolutionary biology...... research. Pinpointing the underlying mechanism of sex ratio bias is challenging owing to the multitude of potential sex ratio-biasing factors. In the dwarf spider, Oedothorax gibbosus, infection with the bacterial endosymbiont Wolbachia results in a female bias. However, pedigree analysis reveals...

  7. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Żmigrodzka, M; Guzera, M; Winnicka, A


    Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

  8. Central neurocytoma : morphological, flow cytometric, polymerase chain reaction, fluorescence in situ hybridization, and karyotypic analyses - Case report

    Jay, RM; Edwards, KA; Hoving, E; Rutka, J; Becker, L; Zielenska, M; Teshima, Teruki


    The results of cytogenetic and molecular genetic analysis of a central neurocytoma are presented. Central neurocytomas are intriguing neoplasms that exhibit primarily neuronal, but also glial characteristics, which indicate an origin from a pluripotential neuroglial precursor. The authors describe a

  9. Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

    Bienenmann-Ploum, M.E.; Vincent, U.; Campbell, K.; Huet, A.C.; Haasnoot, W.; Delahaut, P.; Stolker, A.A.M.; Elliott, C.T.; Nielen, M.W.F.


    Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory valid

  10. Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

    Bienenmann-Ploum, M.E.; Huet, A.C.; Campbell, K.; Fodey, T.L.; Vincent, U.; Delahaut, P.; Elliot, C.T.; Nielen, M.W.F.


    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screen

  11. Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution

    Dalgaard, Tina; Norup, Liselotte Rothmann; Rubbenstroth, Dennis


    fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation...

  12. Immuno-flow cytometric detection of the ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense : Independence of physiological state

    Vrieling, EG; vandePoll, WH; Vriezekolk, G; Gieskes, WWC


    The ichthyotoxic dinoflagellates Gyrodinium aureolum and Gymnodinium nagasakiense were cultured under different environmental conditions to test possible variability in immunochemical labelling intensity of cell-surface antigens using species-specific monoclonal antibodies. Variation of antigen abun

  13. Differential genotoxicity of acrylamide in the micronucleus and Pig-a gene mutation assays in F344 rats and B6C3F1 mice.

    Hobbs, Cheryl A; Davis, Jeffrey; Shepard, Kim; Chepelev, Nikolai; Friedman, Marvin; Marroni, Dennis; Recio, Leslie


    Acrylamide is used in many industrial processes and is present in a variety of fried and baked foods. In rodent carcinogenicity assays, acrylamide exposure leads to tumour formation at doses lower than those demonstrated to induce genotoxic damage. We evaluated the potential of acrylamide to induce structural DNA damage and gene mutations in rodents using highly sensitive flow cytometric analysis of micronucleus and Pig-a mutant frequencies, respectively. Male F344 rats and B6C3F1 mice were administered acrylamide in drinking water for 30 days at doses spanning and exceeding the range of acrylamide exposure tested in cancer bioassays-top dose of 12.0 and 24.0mg/kg/day in mice and in rats, respectively. A positive control, N-ethyl-N-nitrosourea, was administered at the beginning and end of the study to meet the expression time for the two DNA damage phenotypes. The results of the micronucleus and Pig-a assays were negative and equivocal, respectively, for male rats exposed to acrylamide at the concentrations tested. In contrast, acrylamide induced a dose-dependent increase in micronucleus formation but tested negative in the Pig-a assay in mice. Higher plasma concentrations of glycidamide in mice than rats are hypothesized to explain, at least in part, the differences in the response. Benchmark dose modelling indicates that structural DNA damage as opposed to point mutations is most relevant to the genotoxic mode of action of acrylamide-induced carcinogenicity. Moreover, the lack of genotoxicity detected at acrylamide-induced carcinogenicity in rodents. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail:

  14. Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production.

    Byun, J-K; Moon, S-J; Jhun, J-Y; Kim, E-K; Park, J-S; Youn, J; Min, J-K; Park, S-H; Kim, H-Y; Cho, M-L


    Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)(+) follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg ), CD19(+) CD1d(high) CD5(high) , CD19(+) CD25(high) forkhead box protein 3 (FoxP3)(+) regulatory B (Breg ) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS(+) GL-7(+) germinal centre B cells and B220(-) CD138(+) plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4(+) T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA. © 2014 British Society for Immunology.

  15. Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain.

    Matsuo, Hideaki; Uchida, Kenzo; Nakajima, Hideaki; Guerrero, Alexander Rodriguez; Watanabe, Shuji; Takeura, Naoto; Sugita, Daisuke; Shimada, Seiichiro; Nakatsuka, Terumasa; Baba, Hisatoshi


    Although transcutaneous electrical nerve stimulation (TENS) is widely used for the treatment of neuropathic pain, its effectiveness and mechanism of action in reducing neuropathic pain remain uncertain. We investigated the effects of early TENS (starting from the day after surgery) in mice with neuropathic pain, on hyperalgesia, glial cell activation, pain transmission neuron sensitization, expression of proinflammatory cytokines, and opioid receptors in the spinal dorsal horn. Following nerve injury, TENS and behavioral tests were performed every day. Immunohistochemical, immunoblot, and flow cytometric analysis of the lumbar spinal cord were performed after 8 days. Early TENS reduced mechanical and thermal hyperalgesia and decreased the activation of microglia and astrocytes (PEarly TENS decreased p-p38 within microglia (Pearly TENS relieved hyperalgesia in our mouse model of neuropathic pain by inhibiting glial activation, MAP kinase activation, PKC-γ, and p-CREB expression, and proinflammatory cytokines expression, as well as maintenance of spinal opioid receptors. The findings indicate that TENS treatment is more effective when applied as early after nerve injury as possible. Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  16. Supercontinuum white light lasers for flow cytometry

    Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.


    Excitation of fluorescent probes for flow cytometry has traditionally been limited to a few discrete laser lines, an inherent limitation in our ability to excite the vast array of fluorescent probes available for cellular analysis. In this report, we have used a supercontinuum (SC) white light laser as an excitation source for flow cytometry. By selectively filtering the wavelength of interest, almost any laser wavelength in the visible spectrum can be separated and used for flow cytometric analysis. The white light lasers used in this study were integrated into a commercial flow cytometry platform, and a series of high-transmission bandpass filters used to select wavelength ranges from the blue (~480 nm) to the long red (>700 nm). Cells labeled with a variety of fluorescent probes or expressing fluorescent proteins were then analyzed, in comparison with traditional lasers emitting at wavelengths similar to the filtered SC source. Based on a standard sensitivity metric, the white light laser bandwidths produced similar excitation levels to traditional lasers for a wide variety of fluorescent probes and expressible proteins. Sensitivity assessment using fluorescent bead arrays confirmed that the SC laser and traditional sources resulted in similar levels of detection sensitivity. Supercontinuum white light laser sources therefore have the potential to remove a significant barrier in flow cytometric analysis, namely the limitation of excitation wavelengths. Almost any visible wavelength range can be made available for excitation, allowing access to virtually any fluorescent probe, and permitting “fine-tuning” of excitation wavelength to particular probes. PMID:19072836

  17. Brain-derived neurotrophic factor modulates immune reaction in mice with peripheral nerve xenotransplantation

    Yu X


    Full Text Available Xin Yu,1 Laijin Lu,1 Zhigang Liu,1 Teng Yang,2 Xu Gong,1 Yubo Ning,3 Yanfang Jiang4 1Department of Hand Surgery, 2Department of Orthopedics, The First Hospital of Jilin University, Changchun, 3Department of Orthopedics, Ningshi Orthopedics Hospital of Tonghua, Tonghua, 4Department of Central Laboratory, The First Hospital of Jilin University, Changchun, People’s Republic of China Background: Brain-derived neurotrophic factor (BDNF has been demonstrated to play an important role in survival, differentiation, and neurite outgrowth for many types of neurons. This study was designed to identify the role of BDNF during peripheral nerve xenotransplantation. Materials and methods: A peripheral nerve xenotransplantation from rats to mice was performed. Intracellular cytokines were stained for natural killer (NK cells, natural killer T (NKT cells, T cells, and B cells and analyzed by flow cytometry in the spleen of the recipient mouse. Serum levels of related cytokines were quantified by cytometric bead array. Results: Splenic NK cells significantly increased in the xenotransplanted mice (8.47±0.88×107 cells/mL compared to that in the control mice (4.66±0.78×107 cells/mL, P=0.0003, which significantly reduced in the presence of BDNF (4.85±0.87×107 cells/mL, P=0.0004. In contrast, splenic NKT cell number was significantly increased in the mice with xenotransplantation plus BDNF (XT + BDNF compared to that of control group or of mice receiving xenotransplantation only (XT only. Furthermore, the number of CD3+ T cells, CD3+CD4+ T cells, CD3+CD4- T cells, interferon-γ-producing CD3+CD4+ T cells, and interleukin (IL-17-producing CD3+CD4+ T cells, as well as CD3-CD19+ B cells, was significantly higher in the spleen of XT only mice compared to the control mice (P<0.05, which was significantly reduced by BDNF (P<0.05. The number of IL-4-producing CD3+CD4+ T cells and CD3+CD4+CD25+Foxp3+ T cells was significantly higher in the spleen of XT + BDNF

  18. The Effect of Lymphocyte Immunotherapy on CD80+ Cells at the Fetomaternal Interface and Cyesis Result of Mice Model of Spontaneous Abortion

    林羿; 曾耀英; 何贤辉; 曾山; 詹美意; 关洁宾; 狄静芳; 肇静娴; 全世明


    Objectives To explore the relationship between CD80 expression on lymphocytes at the fetomaternal interface and the fertility characteristics in CBA/J × DBA/2 mice as a model of recurrent spontaneous abortion (RSA) and to investigate the effects of lym phocyte immunotherapy (LIT) on the level of CD80 expression.Materials & Methods The characteristics of fertility in CBA/J × DBA/2 mice were observed in a 120-day period and compared with four normal fertile groups. In anoth er 15 pairs of CBA/J × DBA/2 breedings, resorption rate on day 13 of pregnancy were calculated and the proportion of CD80+ cells at the fetomaternal interface were determined by using two-color flow cytometric analysis, mainly stained with CD80 FITC and CD45-PE. In order to determine the identity of CD80+ cells, the expression levels of CD3(T cell marker), DX 5(NK cell marker), and MHC-Ⅱ(antigen present ing cell marker) were detected in this cell population. Furthermore, the resorption rate and the proportion of CD80+ cells among CBA/J × DBA/2 breedings with and with out immunotherapy were also determined and compared with normal fertile controls.Results The characteristics of abortion in CBA/J × DBA/2 mice were recurrent abor tion on about day 10 of gestation. The resorption rate in CBA/J × DBA/2 mice was significantly higher than that in BALB/c×DBA/2 mice (30. 8% ± 16. 6% vs. 7. 7%± 6. 7%, P< 0. 01). Accordingly, the proportion of CD80+ cells evaluated at the fetomaternal interface in CBA/J × DBA/2 mice was also significantly higher (11. 7%± 5. 8% vs. 3. 9% ± 1. 8%, P< 0. 01). Resorption rate of CBA/J × DBA/2 mice un derwent of LIT was significantly lower than that without LIT, and this decreased rate was correlated with decreased proportion of CD80+ cells.Conclusion In CBA/J × DBA/2 mice model, the characteristics of abortion seem to be peri-implantation embryo-resorption. A correlation between early embryonic waste and higher CD80 proportion at the fetomaternal interface

  19. Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice

    Brünner, N; Bronzert, D; Vindeløv, L L


    determined by repeated flow cytometric DNA analyses in vitro and in vivo and by the technique of labeled mitosis in nude mouse-grown tumors. Under in vitro conditions, estradiol induced a pronounced increase in S-phase fraction and cell number. TAM inhibited growth of MCF-7 cells with a concomitant increase...... cytometric DNA analysis and percentage of labeled mitosis investigations revealed no significant differences in the proliferation kinetics of TAM-treated and control tumors. Calculating the cell loss factor demonstrated an increase from 69% in control tumors to 107% in TAM-treated tumors. These experiments...

  20. Changes in color and blood flow of the tongue of HBV transgenic mice%HBV转基因小鼠的舌色改变及血流变化

    刘文兰; 车念聪; 唐佐青; 油红捷; 杨铮


    目的:研究HBV转基因小鼠的舌色表现和微血管血流变化.方法:10只C57BL/6J-HBV转基因小鼠和10只正常小鼠(C57BL/6J非转基因小鼠),均雌雄各半,采用小鼠舌象观察装置对小鼠舌色进行观察;使用激光多普勒血流仪进行舌和肝脏微血管血流检测;断头采血,测定血清丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)的含量;HE染色观察肝和舌组织病理变化.结果:HBV转基因小鼠中,6只小鼠舌色呈紫色,4只舌色呈暗红色,而正常小鼠的舌色均为淡红色.HBV转基因小鼠舌色色调(H值)变深,有显著性差异(P<0.01);舌色亮度(V值)变弱,有极显著性差异(P<0.001).和正常鼠相比,紫舌HBV转基因小鼠微血管血流灌注量和血流速度显著降低(0.206±0.13 vs 0.794±0.13;0.479±0.07 vs 0.331±0.04,P<0.01,P<0.001),该模型肝脏微血管血流灌注量和血流速度显著降低,HBV转基因小鼠肝脏和舌存在明显的炎性改变.结论:HBV转基因小鼠舌色以紫色多见,其形成机制与微循环障碍有关.该模型存在肝脏和舌的炎症病理,提示炎性微环境是形成其肝脏和舌微循环障碍的重要原因.%AIM: To examine the changes in color and microvascular blood flow of the tongue of HBV transgenic mice.METHODS: Ten C57BL/6J-HBV transgenic mice and 10 normal mice were used in the study. The tongue color of the mice was observed daily. The microvascular blood flow of tongue and liver were detected with a laser Doppler blood flowmeter. Blood samples were taken to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Pathological changes in the tongue and liver were evaluated by HE staining.RESULTS: The tongue was purple in color in six HBV transgenic mice and dark red in the remaining four transgenic mice. In contrast,the tongue of normal mice was light red in color. The hue of the tongue become darker and brightness become weaker in HBV transgenic mice (0.206 ± 0

  1. Effect of tamoxifen on the receptor-positive T61 and the receptor-negative T60 human breast carcinomas grown in nude mice

    Brünner, N; Spang-Thomsen, M; Vindeløv, L;


    to a transformed Gompertz function, and the effect on the cell cycle distributions was estimated by flow cytometric DNA analysis on tumour tissue obtained by fine-needle aspirations at intervals after the treatment. The results showed that in the T61 tumour, tamoxifen induced a dose-related growth inhibition...

  2. Growth kinetics and in vivo radiosensitivity in nude mice of two subpopulations derived from a single human small cell carcinoma of the lung

    Spang-Thomsen, M; Clerici, M; Engelholm, S A;


    were described according to a transformed Gompertz function, and the cell kinetics were examined by flow cytometric DNA analysis (FCM) and by the technique of labelled mitoses. The effect of single-dose irradiation was estimated by the specific growth delay calculated from the growth curves...

  3. Preferential Elimination of Older Erythrocytes in Circulation and Depressed Bone Marrow Erythropoietic Activity Contribute to Cadmium Induced Anemia in Mice.

    Chatterjee, Sreoshi; Saxena, Rajiv K


    Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid

  4. Humanized mice dually challenged with R5 and X4 HIV-1 show preferential R5 viremia and restricted X4 infection of CCR5(+)CD4(+) T cells.

    Terahara, Kazutaka; Ishige, Masayuki; Ikeno, Shota; Okada, Seiji; Kobayashi-Ishihara, Mie; Ato, Manabu; Tsunetsugu-Yokota, Yasuko


    CCR5-tropic (R5) immunodeficiency virus type 1 (HIV-1) strains are highly transmissible during the early stage of infection in humans, whereas CXCR4-tropic (X4) strains are less transmissible. This study aimed to explore the basis for early phase R5 and X4 HIV-1 infection in vivo by using humanized mice dually challenged with R5 HIV-1NLAD8-D harboring DsRed and X4 HIV-1(NL-E) harboring EGFP. Whereas R5 HIV-1 replicated well, X4 HIV-1 caused only transient viremia with variable kinetics; however, this was distinct from the low level but persistent viremia observed in mice challenged with X4 HIV-1 alone. Flow cytometric analysis of HIV-1-infected cells revealed that X4 HIV-1 infection of CCR5(+)CD4(+) T cells was significantly suppressed in the presence of R5 HIV-1. X4 HIV-1 was more cytopathic than R5 HIV-1; however, this was not the cause of restricted X4 HIV-1 infection because there were no significant differences in the mortality rates of CCR5(+) and CCR5(-) cells within the X4 HIV-1-infected cell populations. Taken together, these results suggest that restricted infection of CCR5(+)CD4(+) T cells by X4 HIV-1 (occurring via a still-to-be-identified mechanism) might contribute to the preferential transmission of R5 HIV-1 during the early phase of infection.

  5. Chlamydia trachomatis recombinant MOMP encapsulated in PLGA nanoparticles triggers primarily T helper 1 cellular and antibody immune responses in mice: a desirable candidate nanovaccine

    Fairley SJ


    Full Text Available Stacie J Fairley, Shree R Singh, Abebayehu N Yilma, Alain B Waffo, Praseetha Subbarayan, Saurabh Dixit, Murtada A Taha, Chino D Cambridge, Vida A Dennis Center for NanoBiotechnology Research, Alabama State University, Montgomery, AL, USA Abstract: We recently demonstrated by in vitro experiments that PLGA (poly D, L-lactide-co-glycolide potentiates T helper 1 (Th1 immune responses induced by a peptide derived from the recombinant major outer membrane protein (rMOMP of Chlamydia trachomatis, and may be a promising vaccine delivery system. Herein we evaluated the immune-potentiating potential of PLGA by encapsulating the full-length rMOMP (PLGA-rMOMP, characterizing it in vitro, and investigating its immunogenicity in vivo. Our hypothesis was that PLGA-rMOMP triggers Th1 immune responses in mice, which are desirable prerequisites for a C. trachomatis candidate nanovaccine. Physical-structural characterizations of PLGA-rMOMP revealed its size (approximately 272 nm, zeta potential (−14.30 mV, apparent spherical smooth morphology, and continuous slow release pattern. PLGA potentiated the ability of encapsulated rMOMP to trigger production of cytokines and chemokines by mouse J774 macrophages. Flow cytometric analyses revealed that spleen cells from BALB/c mice immunized with PLGA-rMOMP had elevated numbers of CD4+ and CD8+ T cell subsets, and secreted more rMOMP-specific interferon-gamma (Th1 and interleukin (IL-12p40 (Th1/Th17 than IL-4 and IL-10 (Th2 cytokines. PLGA-rMOMP-immunized mice produced higher serum immunoglobulin (IgG and IgG2a (Th1 than IgG1 (Th2 rMOMP-specific antibodies. Notably, sera from PLGA-rMOMP-immunized mice had a 64-fold higher Th1 than Th2 antibody titer, whereas mice immunized with rMOMP in Freund's adjuvant had only a four-fold higher Th1 than Th2 antibody titer, suggesting primarily induction of a Th1 antibody response in PLGA-rMOMP-immunized mice. Our data underscore PLGA as an effective delivery system for a C

  6. Aloe vera (Aloe barbadensis Miller) supplemented probiotic lassi prevents Shigella infiltration from epithelial barrier into systemic blood flow in mice model.

    Hussain, Shaik Abdul; Patil, Girdhari Ramdas; Reddi, Srinu; Yadav, Vidhu; Pothuraju, Ramesh; Singh, Ram Ran Bijoy; Kapila, Suman


    The aim of present work was to investigate preventive role of orally administered Aloe vera supplemented probiotic lassi (APL) on Shigella dysenteriae infection in mice. At the end of experimental period (2, 5 and 7 days of challenging), different organs such as spleen, liver, small intestine, large intestine, and peritoneal fluid were collected and assessed for Shigella colonization. Secretary IgA was estimated in intestinal fluid. Blood was collected in heparinized tubes for various haematological studies. Oral administration of APL showed a significant (p < 0.05) reduction in the Shigella counts (log cfu/mL) in all organs as compared to other treatment groups at different intervals after post feeding. Similarly, secretary IgA antibody levels (μg/mL) in intestinal fluid were significantly (p < 0.05) increased in case of APL fed mice. Further, feeding of APL also demonstrated a positive effect on different haematological parameters viz. Hb (gm %), RBC and WBC count. The results indicated the immunoprotective effects of APL against Shigella dysenteriae induced infection in mice.

  7. Enhanced local and systemic anti-melanoma CD8+ T cell responses after memory T cell-based adoptive immunotherapy in mice

    Contreras, Amanda; Sen, Siddhartha; Tatar, Andrew J.; Mahvi, David A.; Meyers, Justin V.; Srinand, Prakrithi; Suresh, Marulasiddappa


    Adoptive cell transfer (ACT) melanoma immunotherapy typically employs acutely activated effector CD8+ T cells for their ability to rapidly recognize and clear antigen. We have previously observed that effector CD8+ T cells are highly susceptible to melanoma-induced suppression, whereas memory CD8+ T cells are not. Although memory T cells have been presumed to be potentially advantageous for ACT, the kinetics of local and systemic T cell responses after effector and memory ACT have not been compared. B16F10 melanoma cells stably transfected to express very low levels of the lymphocytic choriomeningitis virus (LCMV) peptide GP33 (B16GP33) were inoculated into syngeneic C57BL/6 mice. Equal numbers of bona fide naïve, effector, or memory phenotype GP33-specific CD8+ T cells were adoptively transferred into mice 1 day after B16GP33 inoculation. The efficacy of ACT immunotherapy was kinetically assessed using serial tumor measurements and flow cytometric analyses of local and systemic CD8+ T cell responses. Control of B16GP33 tumor growth, persistence of adoptively transferred CD8+ cells, intratumoral infiltration of CD8+ T cells, and systemic CD8+ T cell responsiveness to GP33 were strongest after ACT of memory CD8+ T cells. Following surgical tumor resection and melanoma tumor challenge, only mice receiving memory T cell-based ACT immunotherapy exhibited durable tumor-specific immunity. These findings demonstrate how the use of non-expanded memory CD8+ T cells may enhance ACT immunotherapeutic efficacy. PMID:27011014

  8. Co-expression of Ubiquitin gene and capsid protein gene enhances the potency of DNA immunization of PCV2 in mice

    Zhou Yanjun


    Full Text Available Abstract A recombinant plasmid that co-expressed ubiquitin and porcine circovirus type 2 (PCV2 virus capsid protein (Cap, denoted as pc-Ub-Cap, and a plasmid encoding PCV2 virus Cap alone, denoted as pc-Cap, were transfected into 293T cells. Indirect immunofluorescence (IIF and confocal microscopy were performed to measure the cellular expression of Cap. Three groups of mice were then vaccinated once every three weeks for a total of three doses with pc-Ub-Cap, pc-Cap or the empty vector pCAGGS, followed by challenging all mice intraperitoneally with 0.5 mL 106.5 TCID50/mL PCV2. To characterize the protective immune response against PCV2 infection in mice, assays of antibody titer (including different IgG isotypes, flow cytometric analysis (FCM, lymphocyte proliferation, cytokine production and viremia were evaluated. The results showed that pc-Ub-Cap and pc-Cap were efficiently expressed in 293T cells. However, pc-Ub-Cap-vaccinated animals had a significantly higher level of Cap-specific antibody and induced a stronger Th1 type cellular immune response than did pc-Cap-vaccinated animals, suggesting that ubiquitin conjugation improved both the cellular and humoral immune responses. Additionally, viral replication in blood was lower in the pc-Ub-Cap-vaccinated group than in the pc-Cap and empty vector groups, suggesting that the protective immunity induced by pc-Ub-Cap is superior to that induced by pc-Cap.

  9. Co-delivery of ccl19 gene enhances anti-caries DNA vaccine pCIA-P immunogenicity in mice by increasing dendritic cell migration to secondary lymphoid tissues

    Yan-hong YAN; Sheng-cai QI; Ling-kai SU; Qing-an XU; Ming-wen FAN


    Aim:To investigate how co-delivery of the gene encoding C-C chemokine ligand-19 (CCL-19) affected the systemic immune responses to an anti-caries DNA vaccine pClA-P in mice.Methods:Plasmid encoding CCL19-GFP fusion protein (pCCL19/GFP) was constructed by inserting murine ccl19 gene into GFPexpressing vector pAcGFP1-N1.Chemotactic effect of the fusion protein on murine dendritic cells (DCs) was assessed in vitro and in vivo using transwell and flow cytometric analysis,respectively.BALB/c mice were administered anti-caries DNA vaccine pClA-P plus pCCL19/GFP (each 100 μg,im) or pClA-P alone.Serum level of anti-PAc IgG was assessed with ELISA.Splenocytes from the mice were stimulated with PAc protein for 48 h,and IFN-y and IL-4 production was measured with ELISA.The presence of pCCL19/GFP in spleen and draining lymph nodes was assessed using PCR.The expression of pCCL19/GFP protein in these tissues was analyzed under microscope and with flow cytometry.Results:The expression level of CCL19-GFP fusion protein was considerably increased 48 h after transfection of C0S-7 cells with pCCL19/GFP plasmids.The fusion protein showed potent chemotactic activity on DCs in vitro.The level of serum PAc-specific IgG was significantly increased from 4 to 14 weeks in the mice vaccinated with pCIA-P plus pCCL19/GFP.Compared to mice vaccinated with pCIA-P alone,the splenocytes from mice vaccinated with pClA-P plus pCCL19/GFP produced significantly higher level of IFN-Y,but IL-4 production had no significant change.Following intromuscular co-delivery,pCCL19/GFP plasmid and fusion protein were detected in the spleen and draining lymph nodes.Administration of CCL19 gene in mice markedly increased the number of mature DCs in secondary lymphoid tissues.Conclusion:CCL19 serves as an effective adjuvant for anti-caries DNA vaccine by inducing chemotactic migration of DCs to secondary lymphoid tissues.

  10. Identification of CD4+ T-cell Epitopes on Mycobacterium Tuberculosis- Secreted MPB51 Protein in C57BL/6 Mice

    A.R. Rafiei


    Full Text Available Introduction & Objective: Both CD4+ type 1 helper (Th1 cells and CD8+ T cells play effective roles in protection against Mycobacterium tuberculosis infection. DNA vaccine encoding MPB51 can induce Th1-type immune responses and protective immunity upon challenge with M.tuberculosis. This study address to identify T-cell immunodominant epitopes on MPB51 in C57BL/6 mice.Materials & Methods : We cloned DNA encoding MPB51 molecule in pCI plasmid. After constructing MPB51 DNA-covered gold cartridge, C57BL/6 mice were immunized by using a gene gun system. Two weeks after the last immunization, the immune spleen cells were cultured in the presence of a synthetic overlapping library peptides covering the mature MPB51 sequence or medium alone. Intracellular and cell culture supernatant gamma interferon (IFN- production was analyzed using flow cytometry and ELISA, respectively.Results : Mapping of T-cell epitopes on MPB51 molecule was performed in the spleen lymphocytes restimulated by 20-mer overlapping synthetic peptides of mature MPB51 sequence. Flow cytometric analysis with intracellular IFN- and the T-cell phenotype revealed that P171-190 and P191-210 peptides contain immunodominant CD4+ T-cell epitopes. Further analysis by using T-cell subset depletion and serial peptide dilution revealed that P171 and p191 are H2-Ab-restricted dominant and subdominant CD4+ T cell epitopes, respectively. Conclusion: This study proved that vaccination with plasmid DNA encoding M. tuberculosis-secreted MPB51 protein not only induce CD4+ T cells immune response but also is an appropriate method for identifying immunogenic peptides.

  11. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

    Oldenburg Delene J


    Full Text Available Abstract Background The amount of DNA in the chloroplasts of some plant species has been shown recently to decline dramatically during leaf development. A high-throughput method of DNA detection in chloroplasts is now needed in order to facilitate the further investigation of this process using large numbers of tissue samples. Results The DNA-binding fluorophores 4',6-diamidino-2-phenylindole (DAPI, SYBR Green I (SG, SYTO 42, and SYTO 45 were assessed for their utility in flow cytometric analysis of DNA in Arabidopsis chloroplasts. Fluorescence microscopy and real-time quantitative PCR (qPCR were used to validate flow cytometry data. We found neither DAPI nor SYTO 45 suitable for flow cytometric analysis of chloroplast DNA (cpDNA content, but did find changes in cpDNA content during development by flow cytometry using SG and SYTO 42. The latter dye provided more sensitive detection, and the results were similar to those from the fluorescence microscopic analysis. Differences in SYTO 42 fluorescence were found to correlate with differences in cpDNA content as determined by qPCR using three primer sets widely spaced across the chloroplast genome, suggesting that the whole genome undergoes copy number reduction during development, rather than selective reduction/degradation of subgenomic regions. Conclusion Flow cytometric analysis of chloroplasts stained with SYTO 42 is a high-throughput method suitable for determining changes in cpDNA content during development and for sorting chloroplasts on the basis of DNA content.

  12. Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls.

    Christensen, Preben; Boelling, Dorothee; Pedersen, Kurt Myrup; Korsgaard, Inge Riis; Jensen, Just


    A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 x 10(6) motile sperm before freezing were used in 118,680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44,946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

  13. Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

    Castellanos-Martínez, S; Prado-Alvarez, M; Lobo-da-Cunha, A; Azevedo, C; Gestal, C


    The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

  14. Monodisperse Water-in-Oil-in-Water (W/O/W Double Emulsion Droplets as Uniform Compartments for High-Throughput Analysis via Flow Cytometry

    Jing Yan


    Full Text Available Here we report the application of monodisperse double emulsion droplets, produced in a single step within partially hydrophilic/partially hydrophobic microfluidic devices, as defined containers for quantitative flow cytometric analysis. Samples with varying fluorophore concentrations were generated, and a clear correlation between dye concentration and fluorescence signals was observed.

  15. A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample

    Mauricas Mykolas


    Full Text Available Abstract Background Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd labelling do not provide information on the cell cycle time (TC and the growth fraction (GF. In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling. Methods The proposed method is based on two assumptions: (1 the number of labelled cells traversing the cell cycle per unit time is constant and (2 the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture. Results The suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 ± 1.1 h and 21.6 ± 0.9 h, respectively. GF in tumours at day 10 was lower than GF at day 4 (54.2 ± 7.7% vs. 79.2 ± 5.9%, p = 0.0003. Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively. Conclusion The proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed

  16. Single antigen flow beads for identification of human leukocyte antigen antibody specificities in hypersensitized patients with chronic renal failure

    Soyöz, Mustafa; Kılıçaslan-Ayna, Tülay; Özkızılcık-Koçyiğit, Aslı; Güleç, Derya; Pirim, İbrahim


    Aims of this study Aims of this study were to identify class I and class II antibodies in highly sensitized patients by flow cytometry single antigen bead (FC-SAB) assay and to evaluate according to donor HLA type in order to increase their kidney transplantation chance. Material and methods We analyzed 60 hypersensitive patients of 351 individuals, who applied to our laboratory for PRA test in November 2013-December 2014. Flow cytometric PRA screening and single antigen bead commercial kits ...

  17. Genetic evidence for p75NTR-dependent tetraploidy in cortical projection neurons from adult mice.

    López-Sánchez, Noelia; Frade, José M


    A subpopulation of chick retinal projection neurons becomes tetraploid during development, an event prevented by blocking antibodies against p75 neurotrophin receptor (p75(NTR)). We have used an optimized flow cytometric assay, based on the analysis of unfixed brain cell nuclei, to study whether p75(NTR)-dependent neuronal tetraploidization takes place in the cerebral cortex, giving rise to projection neurons as well. We show that 3% of neurons in both murine neocortex and chick telencephalic derivatives are tetraploid, and that in the mouse ~85% of these neurons express the immediate early genes Erg-1 and c-Fos, indicating that they are functionally active. Tetraploid cortical neurons (65-80%) express CTIP2, a transcription factor specific for subcortical projection neurons in the mouse neocortex. During the period in which these neurons are born, p75(NTR) is detected in differentiating neurons undergoing DNA replication. Accordingly, p75(NTR)-deficient mice contain a reduced proportion of both NeuN and CTIP2-positive neocortical tetraploid neurons, thus providing genetic evidence for the participation of p75(NTR) in the induction of neuronal tetraploidy in the mouse neocortex. In the striatum tetraploidy is mainly associated with long-range projection neurons as well since ~80% of tetraploid neurons in this structure express calbindin, a marker of neostriatal-matrix spiny neurons, known to establish long-range projections to the substantia nigra and globus pallidus. In contrast, only 20% of tetraploid cortical neurons express calbindin, which is mainly expressed in layers II-III, where CTIP2 is absent. We conclude that tetraploidy mainly affects long-range projection neurons, being facilitated by p75(NTR) in the neocortex.

  18. Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring

    Kline Richard


    Full Text Available Abstract Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. Methods PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining for chemotherapeutic action of different drugs. Results DNA fragmentation, ratiometric ions and fluorescence endlabelling plots were characteristic for cell lines and drug response. 31P-23Na NMR spectra showed characteristic high phospho-choline and sodium peaks. Conclusion Flow cytometry, DNA fragmentation, ion ratiometric methods and NMR peaks indicated apoptosis and offered in vivo drug monitoring method.

  19. Chemosensitivity assay in mice prostate tumor: Preliminary report of flow cytometry, DNA fragmentation, ion ratiometric methods of anti-neoplastic drug monitoring


    Abstract Flow cytometry, DNA fragmentation, ion ratiomateric analysis and NMR peaks characterized drug chemosensitivity of antineoplastic drugs. Hypotheses were: 1. The chemosensitive effect of different cancer cell lines is characteristic; 2. DNA fragmentation, ion ratiometric analysis suggest apoptosis status of tumor cells. Methods PC-3 cell lines were compared with DU-145, LNCaP cell lines in culture for the [Na]i and [Ca]i ion sensing dyes, cell death, NMR peaks and apoptosis staining fo...

  20. Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay.

    Aguedo, Mario; Waché, Yves; Mazoyer, Virginie; Sequeira-Le Grand, Anabelle; Belin, Jean-Marc


    The adhesion of methyl ricinoleate droplets to cells of the yeast Yarrowia lipolytica was investigated. A new cytometric method, relying on the double staining of fatty globules with Nile Red and of cells with Calcofluor, enabled us to quantify methyl ricinoleate droplet adhesion to cells precultured on a hydrophilic or on a hydrophobic carbon source. In this last case, droplet adsorption was enhanced and a MATS (microbial adhesion to solvents) test revealed that this increase was due to Lewis acid-base interactions and not to an increase in the hydrophobic properties of the cell surface. These preliminary results demonstrate that the developed cytometric method is promising for various applications concerning the study of interactions between microorganisms and an emulsified hydrophobic substrates.

  1. Applications of Imaging Flow Cytometry for Microalgae.

    Hildebrand, Mark; Davis, Aubrey; Abbriano, Raffaela; Pugsley, Haley R; Traller, Jesse C; Smith, Sarah R; Shrestha, Roshan P; Cook, Orna; Sánchez-Alvarez, Eva L; Manandhar-Shrestha, Kalpana; Alderete, Benjamin


    The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

  2. Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

    Chilampalli Chandeshwari


    Full Text Available Abstract Background Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. Methods UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Results Magnolol pretreated groups (30, 60 μ g before UVB treatments (30 mJ/cm2, 5 days/week resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose polymerase (PARP, increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705, B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Conclusions Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing

  3. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Iole Macchia; Francesca Urbani; Enrico Proietti


    The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometr...

  4. Adherence and viability of intestinal bacteria to differentiated Caco-2 cells quantified by flow cytometry.

    Grootaert, Charlotte; Boon, Nico; Zeka, Fjoralba; Vanhoecke, Barbara; Bracke, Marc; Verstraete, Willy; Van de Wiele, Tom


    Recent developments in host-microbe research give rise to a growing demand for rapid and accurate methods to quantify bacterial adhesion to epithelial cells. Here, we describe a new flow cytometric method to determine the amount and viability of gut bacteria, adhered to a monolayer of differentiated cells. The latter is a more relevant epithelium model than the suspended eukaryotic cells currently used in flow cytometric protocols. During the development of the method, we monitored the adhesion potential of six bacterial species and an intestinal microbial community to Caco-2 cells. The combination of SYBR Green I/propidium iodide was more efficient than carboxyfluorescein diacetate to stain the bacterial cells. In addition, a better separation between the Caco-2 background signal and viable and dead bacteria was obtained. A precise amount of Triton X-100 was used to detach adhered bacteria from Caco-2 cells and cell debris. Yet, a limited decrease in viability was observed for the intestinal microbial community treated with Triton X-100. The flow cytometric lower detection limit for pure bacterial cultures was 3.0-4.0log/mL, whereas a 5.0-5.5log/mL detection limit was obtained in the presence of Caco-2 cell background. The latter was sufficient to quantify adhered bacteria. To the best of our knowledge, this is the first description of a flow cytometric protocol that quantifies adhesion of both pure and mixed gut microbial cultures to a differentiated monolayer of Caco-2 cells and that allows to distinguish between viable and dead adhered bacteria.

  5. Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

    Huerta, Leonor; Lamoyi, Edmundo; Báez-Saldaña, Armida; Larralde, Carlos


    In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion. Copyright 2002 Wiley-Liss, Inc.

  6. Application of advanced cytometric and molecular technologies to minimal residual disease monitoring

    Leary, James F.; He, Feng; Reece, Lisa M.


    Minimal residual disease monitoring presents a number of theoretical and practical challenges. Recently it has been possible to meet some of these challenges by combining a number of new advanced biotechnologies. To monitor the number of residual tumor cells requires complex cocktails of molecular probes that collectively provide sensitivities of detection on the order of one residual tumor cell per million total cells. Ultra-high-speed, multi parameter flow cytometry is capable of analyzing cells at rates in excess of 100,000 cells/sec. Residual tumor selection marker cocktails can be optimized by use of receiver operating characteristic analysis. New data minimizing techniques when combined with multi variate statistical or neural network classifications of tumor cells can more accurately predict residual tumor cell frequencies. The combination of these techniques can, under at least some circumstances, detect frequencies of tumor cells as low as one cell in a million with an accuracy of over 98 percent correct classification. Detection of mutations in tumor suppressor genes requires insolation of these rare tumor cells and single-cell DNA sequencing. Rare residual tumor cells can be isolated at single cell level by high-resolution single-cell cell sorting. Molecular characterization of tumor suppressor gene mutations can be accomplished using a combination of single- cell polymerase chain reaction amplification of specific gene sequences followed by TA cloning techniques and DNA sequencing. Mutations as small as a single base pair in a tumor suppressor gene of a single sorted tumor cell have been detected using these methods. Using new amplification procedures and DNA micro arrays it should be possible to extend the capabilities shown in this paper to screening of multiple DNA mutations in tumor suppressor and other genes on small numbers of sorted metastatic tumor cells.

  7. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

    Salles, Isabelle I; Thijs, Tim; Brunaud, Christine; De Meyer, Simon F; Thys, Johan; Vanhoorelbeke, Karen; Deckmyn, Hans


    Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

  8. Pharmacodynamic Studies on Rabdosia japonica with Myocardial Blood Flow Perfusion in Mice%蓝萼香茶菜增加小鼠心肌血流灌注量的药效动力学

    任常顺; 朱晓红; 王强


    Objective To investigate the pharmacodynamics of Rabdosia japonica.Methods The myocardial blood flow perfusion in mice was measured.Results There was on simple compartment model when Rabdosia japonica was administrated orally in the mouse.The minimal effective·dose was 0.7899 g ·kg-1.The duration and peak time of effective action was 6 h and 1 h respectively.Conclusion Rabdosia japonica can be characterized by fast absorption,slow elimination and a long effective action time.%目的 探讨蓝萼香茶菜药效动力学.方法 以小鼠心肌血流灌注量为指标进行测定.结果 蓝萼香茶菜在小鼠体内并非呈简单的房室模型,其最低起效剂量为生药0.7899 g ·kg-1,效应达峰时间约为1h,效应作用期长达6h以上.结论 蓝萼香茶菜体内具有吸收快,消除慢和作用维持时间长等特点.

  9. Trichosanthin inhibits breast cancer cell proliferation in both cell lines and nude mice by promotion of apoptosis.

    Evandro Fei Fang

    Full Text Available Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS is a ribosome inactivating protein purified from tubers of the Chinese herbal plant Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen. In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.

  10. CD4+CD25+ regulatory T cell depletion modulates anxiety and depression-like behaviors in mice.

    Soo-Jeong Kim

    Full Text Available Stress has been shown to suppress immune function and increase susceptibility to inflammatory disease and psychiatric disease. CD4(+CD25(+ regulatory T (Treg cells are prominent in immune regulation. This study was conducted to determine if anti-CD25 antibody (Ab mediated depletion of Treg cells in mice susceptibility to stress-induced development of depression-like behaviors, as well as immunological and neurochemical activity. To accomplish this, an elevated plus-maze test (EPM, tail suspension test (TST, and forced swim test (FST were used to examine depression-like behaviors upon chronic immobilization stress. Immune imbalance status was observed based on analysis of serum cytokines using a mouse cytometric bead array in conjunction with flow cytometry and changes in the levels of serotonin (5-HT and dopamine (DA in the brain were measured by high performance liquid chromatography (HPLC. The time spent in the open arms of the EPM decreased significantly and the immobility time in the FST increased significantly in the anti-CD25 Ab-treated group when compared with the non stressed wild-type group. In addition, interlukin-6 (IL-6, tumor necrosis factor-á (TNF-á, interlukin-2 (IL-2, interferon-gamma (IFN-γ, interlukin-4 (IL-4 and interlukin-17A (IL-17A concentrations were significantly upregulated in the stressed anti-CD25 Ab-treated group when compared with the non stressed wild-type group. Furthermore, the non stressed anti-CD25 Ab-treated group displayed decreased 5-HT levels within the hippocampus when compared with the non stressed wild-type group. These results suggest that CD4(+CD25(+ Treg cell depletion modulated alterations in depressive behavior, cytokine and monoaminergic activity. Therefore, controlling CD4(+CD25(+ Treg cell function during stress may be a potent therapeutic strategy for the treatment of depression-like symptoms.

  11. A simple and rapid flow cytometry-based assay to identify a competent embryo prior to embryo transfer

    Pallinger, Eva; Bognar, Zoltan; Bodis, Jozsef; Csabai, Timea; Farkas, Nelli; Godony, Krisztina; Varnagy, Akos; Buzas, Edit; Szekeres-Bartho, Julia


    Multiple pregnancy is a risk for prematurity and preterm birth. The goal of assisted reproduction is to achieve a single pregnancy, by transferring a single embryo. This requires improved methods to identify the competent embryo. Here, we describe such a test, based on flow cytometric determination of the nucleic acid (PI+) containing extracellular vesicle (EV) count in day 5 embryo culture media. 88 women undergoing IVF were included in the study. More than 1 embryos were transferred to most...

  12. Identification of inflammatory cells in bovine milk by flow cytometry.

    Redelman, D; Butler, S; Robison, J; Garner, D


    Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.

  13. The n-hexane and chloroform fractions of Piper betle L. trigger different arms of immune responses in BALB/c mice and exhibit antifilarial activity against human lymphatic filarid Brugia malayi.

    Singh, Meghna; Shakya, Shilpy; Soni, Vishal Kumar; Dangi, Anil; Kumar, Nikhil; Bhattacharya, Shailja-Misra


    Modulation of immune functions by using herbal plants and their products has become fundamental regime of therapeutic approach. Piper betle Linn. (Piperaceae) is a widely distributed plant in the tropical and subtropical regions of the world and has been attributed as traditional herbal remedy for many diseases. We have recently reported the antifilarial and antileishmanial efficacy in the leaf extract of Bangla Mahoba landrace of P. betle which is a female plant. The present report describes the in vivo immunomodulatory efficacy of the crude methanolic extract and its n-hexane, chloroform, n-butanol fractions of the female plant at various dose levels ranging between 0.3 and 500 mg/kg in BALB/c. Attempts were also made to observe antifilarial activity of the active extracts and correlate it with the antigen specific immune responses in another rodent Mastomys coucha infected with human lymphatic filarial parasite Brugia malayi. The crude methanol extract and n-hexane fraction were found to potentiate significant (p<0.001) enhancement of both humoral (plaque forming cells, hemagglutination titre) as well as cell-mediated (lymphoproliferation, macrophage activation, delayed type hypersensitivity) immune responses in mice. The flow cytometric analysis of splenocytes of treated mice indicated enhanced population of T-cells (CD4(+), CD8(+)) and B-cells (CD19(+)). The n-hexane fraction (3 mg/kg) was found to induce biased type 2 cytokine response as revealed by increased IL-4(+) and decreased IFN-gamma(+) T-cell population while the chloroform fraction (10 mg/kg) produced a predominant type 1 cytokines. Crude methanolic extract (100 mg/kg) demonstrated a mixed type 1 and type 2 cytokine responses thus suggesting a remarkable immunomodulatory property in this plant. The induction of differential T-helper cell immune response appears ideal to overcome immunosuppression as observed in case of lymphatic, filarial Brugia malayi infection which may also be extended to other

  14. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

    Verthé, Kristof; Verstraete, Willy


    In this study, the use of flow cytometry to analyze phage-mediated killing of Enterobacter aerogenes under varying conditions of temperature and nutrient availability was assessed. Bacteriophage UZ1, specific for an E. aerogenes strain, was applied at a multiplicity of infection (MOI) of 1 and 1000 to a Teflon surface, artificially infected with its host at a level of 4.5 log cells. After incubation for 20 h, bacteriophages were quantified using the soft agar layer method. For the quantification of bacterial cells, plate counting and flow cytometric analysis of live/dead stained cells were performed in parallel. At an MOI of 1, phage treatment was successful only after incubation under nutrient-rich conditions at 37 degrees C: E. aerogenes cells were not detected and a tenfold increase in phage UZ1 was observed. At a MOI of 1000, no E. aerogenes cells could be cultured after incubation at 37 and 4 degrees C. However, flow cytometric analysis revealed that lysis did not occur at 4 degrees C but was achieved during subsequent plate culture. In conclusion, the use of flow cytometry enabled identification of culture-based bias during plate culture. The flow cytometric assay used in this study proved to be rapid, as this culture-independent method does not require lengthy incubation periods post-sampling. The bacteriophage-mediated killing of E. aerogenes cells on Teflon surfaces indicated that disinfection of E. aerogenes with bacteriophage UZ1 can be successful when high MOIs are achieved, while at low multiplicities of infection conditions favorable for phage replication are required.

  15. Flow cytometric analysis of cytokine expression in short-term allergen-stimulated T cells mirrors the phenotype of proliferating T cells in long-term cultures

    Van Hemelen, D.; Elberink, J. N. G. Oude; Bohle, B.; Heimweg, J.; Nawijn, M. C.; van Oosterhout, A. J. M.


    Background: Allergen-specific T(H) cells play an important role in IgE-mediated disorders as allergies. Since this T(H) cell-population only accounts for a small percentage of Tv, cells, they are difficult to phenotype without prior selection or expansion. Methods: Grass-pollen-specific T(H) cell pr



    Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows

  17. Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

    Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.


    The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

  18. Enhanced binding of zona pellucida proteins to the acrosomal region of intact boar spermatozoa in response to fertilizing conditions: a flow cytometric study

    Harkema, W.; Harrison, R.A.P.; Miller, N.G.A.; Topper, E.K.; Woelders, H.


    In this investigation we sought to determine whether sperm capacitation in vitro is accompanied by changes in the functional presence of zona binding sites on the plasma membrane of boar spermatozoa. During sperm incubation at 39°C in various modifications of a Tyrode's-based in vitro fertilization

  19. Two-Color Flow Cytometric Analysis of Intraerythrocytic Malaria Parasite DNA and Surface Membrane-Associated Antigen in Erythrocytes Infected with Plasmodium falciparum


    Infected erythrocytes were fixea with 0.025% glutaraidehyde, followed by treatment with 1%saponiu to gain acceiss to intramembranous components and...Antigen in Erythrocytes Infected With Plasmodium falciparum 1 Kovit Pattanapanyasat,2 Rachanee Udomsangpetch, and H. Kyle Webster The Thalassemia Center...glutaral- izonts. Simultaneous measurement of dehyde followed by treatment with 1% parasite DNA and antigen in the infected saponin to gain access to

  20. Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis.

    Verhoeven, Harrie A; van Griensven, Leo J L D


    Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed decreased polarization and low ROS increase; MDA-MB-435S had limited depolarization and no ROS increase. THP-1 cells exposed to a range of 3BP concentrations in combination with DCA showed increase of polarization, slight ROS increase, and weakened nuclear integrity. 3BP and DCA show no synergism, but have complementary destructive effects on THP-1 cells. The data led to the conclusion that the THP-1 cells do not carry a functional membrane monocarboxylate transporter (MCT) or that 3BP circumvents MCT binding and can enter these cells independently.

  1. Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis

    Verhoeven, H.A.; Griensven, van L.J.L.D.


    Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed

  2. Lymphocyte profiles in multiple myeloma and monoclonal gammopathy of undetermined significance: flow-cytometric characterization and analysis in a two-dimensional correlation biplot.

    Van den Hove, L E; Meeus, P; Derom, A; Demuynck, H; Verhoef, G E; Vandenberghe, P; Boogaerts, M A


    The distribution of 27 T-, B-, and natural killer-cell subsets in the peripheral blood of 40 patients with multiple myeloma (MM), ten patients with monoclonal gammopathy of undetermined significance (MGUS), and 40 healthy donors was investigated by means of classical univariate statistics and advanced multivariate data-analytical techniques. The latter approach was used to describe, represent, and analyze lymphocyte subset distribution in a two-dimensional correlation biplot, allowing comparison of complex lymphocyte profiles (i.e., compound lymphocyte subset distributions) of individual subjects rather than isolated subset values of selected patient and/or donor groups. The correlation biplot revealed that, in accordance with the univariate statistics, the MM patients were characterized by marked shifts towards CD8+, CD57+, CD62L-, CD(16+56)+, and HLA-DR+ T cells, suggesting in vivo immune activation. The activation profile was most markedly observed in treated MM patients in the advanced disease stage category. The lymphocyte profiles of MGUS patients were heterogeneous, with approximately half of them located in the swarm of MM patients and the other half in the swarm of healthy donors. Although the univariate statistics revealed significant differences between MGUS patients and healthy donors only within the B-cell compartment, the correlation biplot revealed that two MGUS patients clearly had a typical T-cell activation profile similar to that of the MM patients. One MGUS patient with a T-cell activation profile progressed 13 months later to a stage IA MM and required chemotherapy. A marked lymphocyte profile shift in one MM patient was associated with terminal and aggressive disease transformation. Our study illustrates further the practical use of correlation biplots for the detection of aberrant lymphocyte profiles and/or profile shifts in individual patients.

  3. Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b.

    Pickering, Jerry W; Martins, Thomas B; Schroder, M Carl; Hill, Harry R


    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

  4. Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b

    Pickering, Jerry W.; Martins, Thomas B.; Schroder, M. Carl; Hill, Harry R.


    We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays ...

  5. Flow cytometric analysis of lymphocytes in aplastic anemia among atomic bomb survivors. With special reference to immunological mechanisms and bolus methylprednisolone therapy

    Imamura, Nobutaka; Inada, Tominari; Asaoku, Hideki; Abe, Kazuhiro; Oguma, Nobuo; Kuramoto, Atsushi


    In 6 patients with aplastic anemia and 3 patients with pernicious anemia, lymphocyte subpopulations in the peripheral blood were measured, before and after steroid therapy, with a fluorescence-activated cell sorder using various monoclonal antibodies. The ratio of OKT4-positive lymphocytes (T4) to OKT8-positive lymphocytes (T8) in the peripheral blood was reduced in 2 patients (20%). The T4/T8 ratio returned to normal during remission of anemia. Hematological improvement was seen after a large amount of steroid therapy in 3 patients. The number of Tac-positive cells tended to decrease and the T4/T8 ratio tended to return to normal with hematological improvement, although there was no correlation to hydrocortisone reaction. Some patients were supposed to have abnormal number of suppressor and inducer T cells. (Namekawa, K.).




    Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows

  7. Flow cytometric detection of growth factor receptors in autografts and analysis of growth factor concentrations in autologous stem cell transplantation: possible significance for platelet recovery

    Schiødt, I; Jensen, Charlotte Harken; Kjaersgaard, E


    In order to improve prediction of hematopoietic recovery, we conducted a pilot study, analyzing the significance of growth factor receptor expression in autografts as well as endogenous growth factor levels in blood before, during and after stem cell transplantation. Three early acting (stem cell...

  8. Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

    Manti, Anita; Boi, Paola; Amalfitano, Stefano; Puddu, Alberto; Papa, Stefano


    Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.

  9. Oral delivery of the Sj23LHD-GST antigen by Salmonella typhimurium type III secretion system protects against Schistosoma japonicum infection in mice.

    Guo Chen


    Full Text Available BACKGROUND: Schistosomiasis japonica is a zoonotic parasitic disease and oral vaccine delivery system would be benefit for prevention of this disease. Although attenuated salmonella has been used as an antigen expression vector for oral vaccine development, the membrane-bound vacuoles in which bacteria reside hinders the presentation of expressed heterologous antigens to the major histocompatibility complex (MHC molecules. The present work used an attenuated Salmonella typhimurium strain VNP20009 to secretory expression of Sj23LHDGST bivalent antigen from Schistosoma japonicum and tested the protective efficacy against S. japonicum infection in orally immunized mice. METHODOLOGY/PRINCIPAL FINDINGS: Promoters (nirB or pagC were used to express the antigen (Sj23LHDGST and the Salmonella type III or α-hemolysin secretion system was employed to secrete it. The immunoblotting analysis and fluorescent microscopy revealed that the antigen was effectively expressed and delivered to the cytosol of macrophages in vitro. Among recombinant vaccine strains, an engineered VNP20009 which expressed the antigen by nirB promoter and secreted it through type III secretion system (nirB-sopE(1-104-Sj23LHD-GST efficiently protected against S. japonicum infection in a mouse model. This strain elicited a predominantly IgG(2a antibody response and a markedly increase in the production of IL-12 and IFN-γ. The flow cytometric analysis demonstrated that this strain caused T cell activation as evidenced by significantly increased expression of CD44 and CD69. CONCLUSION/SIGNIFICANCE: Oral delivery of antigen by nirB-driven Salmonella typhimurium type III secretion system is a novel, safe, inexpensive, efficient and convenient approach for schistosome vaccine development.

  10. Single channel layer, single sheath-flow inlet microfluidic flow cytometer with three-dimensional hydrodynamic focusing.

    Lin, Shiang-Chi; Yen, Pei-Wen; Peng, Chien-Chung; Tung, Yi-Chung


    Flow cytometry is a technique capable of optically characterizing biological particles in a high-throughput manner. In flow cytometry, three dimensional (3D) hydrodynamic focusing is critical for accurate and consistent measurements. Due to the advantages of microfluidic techniques, a number of microfluidic flow cytometers with 3D hydrodynamic focusing have been developed in recent decades. However, the existing devices consist of multiple layers of microfluidic channels and tedious fluidic interconnections. As a result, these devices often require complicated fabrication and professional operation. Consequently, the development of a robust and reliable microfluidic flow cytometer for practical biological applications is desired. This paper develops a microfluidic device with a single channel layer and single sheath-flow inlet capable of achieving 3D hydrodynamic focusing for flow cytometry. The sheath-flow stream is introduced perpendicular to the microfluidic channel to encircle the sample flow. In this paper, the flow fields are simulated using a computational fluidic dynamic (CFD) software, and the results show that the 3D hydrodynamic focusing can be successfully formed in the designed microfluidic device under proper flow conditions. The developed device is further characterized experimentally. First, confocal microscopy is exploited to investigate the flow fields. The resultant Z-stack confocal images show the cross-sectional view of 3D hydrodynamic with flow conditions that agree with the simulated ones. Furthermore, the flow cytometric detections of fluorescence beads are performed using the developed device with various flow rate combinations. The measurement results demonstrate that the device can achieve great detection performances, which are comparable to the conventional flow cytometer. In addition, the enumeration of fluorescence-labelled cells is also performed to show its practicality for biological applications. Consequently, the microfluidic

  11. EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols.

    Kalina, T; Flores-Montero, J; van der Velden, V H J; Martin-Ayuso, M; Böttcher, S; Ritgen, M; Almeida, J; Lhermitte, L; Asnafi, V; Mendonça, A; de Tute, R; Cullen, M; Sedek, L; Vidriales, M B; Pérez, J J; te Marvelde, J G; Mejstrikova, E; Hrusak, O; Szczepański, T; van Dongen, J J M; Orfao, A


    The EU-supported EuroFlow Consortium aimed at innovation and standardization of immunophenotyping for diagnosis and classification of hematological malignancies by introducing 8-color flow cytometry with fully standardized laboratory procedures and antibody panels in order to achieve maximally comparable results among different laboratories. This required the selection of optimal combinations of compatible fluorochromes and the design and evaluation of adequate standard operating procedures (SOPs) for instrument setup, fluorescence compensation and sample preparation. Additionally, we developed software tools for the evaluation of individual antibody reagents and antibody panels. Each section describes what has been evaluated experimentally versus adopted based on existing data and experience. Multicentric evaluation demonstrated high levels of reproducibility based on strict implementation of the EuroFlow SOPs and antibody panels. Overall, the 6 years of extensive collaborative experiments and the analysis of hundreds of cell samples of patients and healthy controls in the EuroFlow centers have provided for the first time laboratory protocols and software tools for fully standardized 8-color flow cytometric immunophenotyping of normal and malignant leukocytes in bone marrow and blood; this has yielded highly comparable data sets, which can be integrated in a single database.

  12. Effects of methylprednisolone on concanavalin A-induced human lymphocyte blastogenesis: a comparative analysis by flow cytometry, volume determination and /sup 3/H-thymidine incorporation

    Marder, P.; Schmidtke, J.R.


    The inhibition of concanavalin A-induced human peripheral blood lymphocyte blastogenesis by methylprednisolone (MP) was studied by using flow cytometry and tritiated thymidine (/sup 3/H-TdR) incorporation. Flow cytometric determinations of volume, low angle forward light scatter, and nucleic acid showed MP to be a potent inhibitor of blastogenesis. The effects were concentration-dependent and correlated with /sup 3/H-TdR uptake. By using the single cell analytic capability of flow cytometry, the target stages of the cell cycle where MP affects lymphocyte activation were determined. Evidence is presented that steroids can block both early and late phases of this process.

  13. Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry.

    de Guise, S; Flipo, D; Boehm, J R; Martineau, D; Béland, P; Fournier, M


    Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

  14. Effect of Benzene on Proliferation and Apoptosis of Splenic Lymphocytes in Mother Generation and Offspring Mice%苯对母鼠和子鼠脾淋巴细胞的增殖与凋亡影响

    旷亦乐; 李纯颖; 杨双波; 李紫; 吴成秋


    Objective To explore the effect of benzene on proliferation and apoptceis of splenic lymphocytes in mother generation and offspring mice. Methods Forty pregnant mice were divided averagely into 4 groups at random. From the 7th day after pregnancy, each of group was exposed to benzene vapour until to parturition (0.0, 5.0, 10.0 and 15.0 mg/m3,respectively), 2 hours par day. At the 1st and 7th days after parturition, 5 mother generation mice end 5 offspring mice of each group were killed to detect the proliferation, cell cycle and apoptosis of splenic lymphocytes in mother generation and offspring mice by MTT assay and flow cytometric analysis. Results During the 1st day and 7th day after parturition, the proliferation of splenic lymphocytes of mother generation and offspring mice in the middle - and high - concentration of benzene groups was inhibited significantly in a concentration-dependent manner (P<O. 05). The cell cycle of splenic lymphocytes of mother generation and offspring mice was blocked in G0/G1 phase in the middle - and high - concentration of benzene groups. The quantity of splenic lymphocytes apoptosis was increased significantly in a concentration- dependent manner in each benzene group (P < 0.05). Conclusion Benzene exposure during pregnancy can damage the immunological function of mother generation mice and offspring mice.%目的 探讨妊娠期接触苯对母鼠及其子鼠免疫功能的影响.方法 40只孕鼠被随机等分为空气对照组和5.0、10.0、15.0 mg/m3三个不同浓度的苯染毒组,各组从孕7 d开始,连续染毒至分娩,每天染毒2 h.分别在分娩后的1 d及7 d,每组取5只母鼠和5只子鼠处死,取脾制备脾淋巴细胞;检测母鼠和子鼠脾淋巴细胞增殖力、细胞周期及细胞凋亡.结果 在分娩后1 d及7 d,中、高浓度苯染毒组母鼠及子鼠的脾淋巴细胞增殖力均低于对照组(P<0.05),并有明显的剂量-效应关系(P<0.05);子鼠与母鼠的淋巴细胞增殖抑制

  15. Digital analysis and sorting of fluorescence lifetime by flow cytometry.

    Houston, Jessica P; Naivar, Mark A; Freyer, James P


    Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 micros, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to >2 ns, corresponding to errors in actual phase shifts from 0.0142 degrees to 1.6 degrees. The lowest coefficients of variation (digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to approximately 98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better

  16. Analysis of Nuclear DNA Content in Capsicum (Solanaceae) by Flow Cytometry and Feulgen Densitometry

    MOSCONE, EDUARDO A.; BARANYI, MONIKA; EBERT, IRMA; Greilhuber, Johann; Ehrendorfer, Friedrich; Hunziker, Armando T.


    Flow cytometric measurements of nuclear DNA content were performed using ethidium bromide as the DNA stain (internal standard, Hordeum vulgare ‘Ditta’, 1C = 5·063 pg) in 25 samples belonging to nine diploid species and four varieties of Capsicum: C. chacoense, C. parvifolium, C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum var. baccatum, C. baccatum var. pendulum, C. baccatum var. umbilicatum, C. eximium and C. pubescens, all with 2n = 24, and C. campylopodium with 2n = 26. In ...

  17. Microfluidic MEMS hand-held flow cytometer

    Grafton, Meggie M. G.; Maleki, Teimour; Zordan, Michael D.; Reece, Lisa M.; Byrnes, Ron; Jones, Alan; Todd, Paul; Leary, James F.


    Due to a number of recent technological advances, a hand-held flow cytometer can be achieved by use of semiconductor illuminators, optical sensors (all battery powered) and sensitive cell markers such as immuno-quantum dot (Qdot) labels. The specific application described is of a handheld blood analyzer that can quickly process a drop of whole, unfractionated human peripheral blood by real-time, on-chip magnetic separation of white blood cells (WBCs) and red blood cells (RBCs) and further fluorescence analysis of Qdot labeled WBC subsets. Various microfluidic patterns were fabricated in PDMS and used to characterize flow of single cells and magnetic deflection of magnetically labeled cells. An LED excitation, avalanche photodiode detection system (SensL Technologies, Ltd., Cork, Ireland) was used for immuno-Qdot detection of WBC subsets. A static optical setup was used to determine the sensitivity of the detection system. In this work we demonstrate: valve-less, on-chip magnetic sorting of immunomagnetically labeled white blood cells, bright Qdot labeling of lymphocytes, and counting of labeled white blood cells. Comparisons of these results with conventional flow cytometric analyses are reported. Sample preparation efficiency was determined by labeling of isolated white blood cells. Appropriate flow rates were determined for optical detection and confirmed with flowing particles. Several enabling technologies required for a truly portable, battery powered, hand-held flow cytometer for use in future point-of-care diagnostic devices have been demonstrated. The combining of these technologies into an integrated handheld instrument is in progress and results on whole blood cell analysis are to be reported in another paper.

  18. Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

    Chan, Shih-Feng; Chen, Ya-Yin; Lin, Jen-Jyh; Liao, Ching-Lung; Ko, Yang-Ching; Tang, Nou-Ying; Kuo, Chao-Lin; Liu, Kuo-Ching; Chung, Jing-Gung


    Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca(2+) release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide

  19. Dynamic proliferation assessment in flow cytometry.

    Diermeier-Daucher, Simone; Brockhoff, Gero


    Dynamic proliferation assessment via flow cytometry is legitimately supposed to be the most powerful tool for recording cell cycle kinetics in-vitro. The preeminent feature is a single cell-based multi-informative analysis by temporal high-resolution. Flow cytometric approaches are based on labeling of proliferating cells via thymidine substitution by a base analog (e.g., 5-bromo-2'-deoxyuridine, BrdU) that is added to cell cultures either for a short period of time (pulse labeling) or continuously until cell harvesting. This unit describes the alternative use of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) in place of BrdU for three different applications: (1) dynamic proliferation assessment by EdU pulse cell labeling; (2) the same approach as (1) but in combination with live/dead cell discrimination; and (3) dynamic cell cycle analysis based on continuous cell labeling with EdU and Hoechst fluorochrome quenching. In contrast to the detection of BrdU incorporation, EdU-positive cells can be identified by taking advantage of click chemistry, which facilitates a simplified and fast cell preparation. Further analysis options but also limitations of the utilization of EdU are discussed.

  20. Flow visualization

    Merzkirch, Wolfgang


    Flow Visualization describes the most widely used methods for visualizing flows. Flow visualization evaluates certain properties of a flow field directly accessible to visual perception. Organized into five chapters, this book first presents the methods that create a visible flow pattern that could be investigated by visual inspection, such as simple dye and density-sensitive visualization methods. It then deals with the application of electron beams and streaming birefringence. Optical methods for compressible flows, hydraulic analogy, and high-speed photography are discussed in other cha

  1. Flow cytometry-based DNA hybridization and polymorphism analysis

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.


    Functional analysis of the humane genome, including the quantification of differential gene expression and the identification of polymorphic sites and disease genes, is an important element of the Human Genome Project. Current methods of analysis are mainly gel-based assays that are not well-suited to rapid genome-scale analyses. To analyze DNA sequence on a large scale, robust and high throughput assays are needed. The authors are developing a suite of microsphere-based approaches employing fluorescence detection to screen and analyze genomic sequence. The approaches include competitive DNA hybridization to measure DNA or RNA targets in unknown samples, and oligo ligation or extension assays to analyze single-nucleotide polymorphisms. Apart from the advances of sensitivity, simplicity, and low sample consumption, these flow cytometric approaches have the potential for high throughput multiplexed analysis using multicolored microspheres and automated sample handling.

  2. Construction of BAC Libraries from Flow-Sorted Chromosomes.

    Šafář, Jan; Šimková, Hana; Doležel, Jaroslav


    Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

  3. Reference values of fetal erythrocytes in maternal blood during pregnancy established using flow cytometry.

    de Wit, Harry; Nabbe, Karin C A M; Kooren, Jurgen A; Adriaansen, Henk J; Roelandse-Koop, Elianne A; Schuitemaker, Joost H N; Hoffmann, Johannes J M L


    The aim of our study was to assess the fetal RBC count in maternal blood during uncomplicated pregnancies from 26 weeks onward. We used a flow cytometric method specifically designed for use in a routine hematology analyzer. Pregnant women were recruited through midwives. The participating laboratories used the FMH QuikQuant method (Trillium Diagnostics, Brewer, ME) in a CELL-DYN Sapphire hematology analyzer (Abbott Diagnostics, Santa Clara, CA). The method is based on a monoclonal antibody to hemoglobin F. Flow cytometric data were analyzed by 2 independent observers. The 95th percentile reference range was estimated according to Clinical and Laboratory Standards Institute guidelines. A total of 236 samples were statistically analyzed. Gestational ages ranged from 21.6 to 41 weeks (mean, 32.0 weeks), and the fetal RBC count in maternal blood ranged from 0.00% to 0.50% (median, 0.025%). The fetal RBC count in maternal blood shows no correlation with gestational age. The established reference range during normal pregnancy is less than 0.125%.

  4. A New Submersible Imaging-in-flow Instrument to Monitor Nano- and Microplankton: Imaging FlowCytobot

    Olson, R. J.; Sosik, H. M.; Shalapyonok, A.


    Understanding of how coastal plankton communities are regulated has traditionally been limited by undersampling, but cabled observatories now provide opportunities to deploy submersible sensors that have high power and data transmission requirements. We have developed an in situ instrument to carry out high-resolution, long term monitoring of phytoplankton and microzooplankton in the size range 10 to100 micrometers, to be deployed at cabled research facilities such as the Martha's Vineyard Coastal Observatory (MVCO). The new instrument is designed to complement FlowCytobot, a submersible flow cytometer currently deployed at MVCO that uses fluorescence and light scattering signals from a laser beam to characterize the smallest phytoplankton cells (less than 10 micrometers). Imaging FlowCytobot uses a combination of flow cytometric and video technology to capture images of organisms for identification and to measure chlorophyll fluorescence associated with each image. Images will be classified using neural net software, while the measurements of chlorophyll fluorescence will allow us to discriminate heterotrophic from phototrophic cells. The new instrument, like the original FlowCytobot is autonomous but remotely programmable. It utilizes a computer controlled syringe pump and distribution valve that allows periodic anti-fouling treatment and analysis of standard beads. Samples are analyzed continuously (0.25 to 2.5 ml per min) and data is sent over a fiber optic link to a remote computer for analysis. Preliminary results indicate that we can detect cells as small as 5 micrometers and discriminate several taxa of diatoms and dinoflagellates.

  5. Chromosomes in the flow to simplify genome analysis.

    Doležel, Jaroslav; Vrána, Jan; Safář, Jan; Bartoš, Jan; Kubaláková, Marie; Simková, Hana


    Nuclear genomes of human, animals, and plants are organized into subunits called chromosomes. When isolated into aqueous suspension, mitotic chromosomes can be classified using flow cytometry according to light scatter and fluorescence parameters. Chromosomes of interest can be purified by flow sorting if they can be resolved from other chromosomes in a karyotype. The analysis and sorting are carried out at rates of 10(2)-10(4) chromosomes per second, and for complex genomes such as wheat the flow sorting technology has been ground-breaking in reducing genome complexity for genome sequencing. The high sample rate provides an attractive approach for karyotype analysis (flow karyotyping) and the purification of chromosomes in large numbers. In characterizing the chromosome complement of an organism, the high number that can be studied using flow cytometry allows for a statistically accurate analysis. Chromosome sorting plays a particularly important role in the analysis of nuclear genome structure and the analysis of particular and aberrant chromosomes. Other attractive but not well-explored features include the analysis of chromosomal proteins, chromosome ultrastructure, and high-resolution mapping using FISH. Recent results demonstrate that chromosome flow sorting can be coupled seamlessly with DNA array and next-generation sequencing technologies for high-throughput analyses. The main advantages are targeting the analysis to a genome region of interest and a significant reduction in sample complexity. As flow sorters can also sort single copies of chromosomes, shotgun sequencing DNA amplified from them enables the production of haplotype-resolved genome sequences. This review explains the principles of flow cytometric chromosome analysis and sorting (flow cytogenetics), discusses the major uses of this technology in genome analysis, and outlines future directions.

  6. Determination of the viability of Aeromonas hydrophila in different types of water by flow cytometry, and comparison with classical methods.

    Pianetti, Anna; Falcioni, Tania; Bruscolini, Francesca; Sabatini, Luigia; Sisti, Elivio; Papa, Stefano


    The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.



    @@ The MICE (Meetings, Incentives, Conventions, and Exhibitions) industry has exploded worldwide over the past decade. The benefits brought by meetings, incentives, conventions, and exhibitions are also benefiting other sectors involved in MICE events, including hotels, travel, and retail. Industry analysts estimate that the income from the global MICE industry will soon exceed USD 220 billion, and is expected to increase by 8-10% each year.

  8. Flow Control


    an aerodynamic design. A few examples of this type of flow control are winglets , fins, or dimples on a golf ball. The other type of flow control is...represented the density states of the flow field. The first parameter was the composition of the regression vector, Θ j. This regression vector was...Development Using Proper Orthogonal De- composition and Volterra Theory. In AIAA 2003-1922, 2003. A. Mani, M. Wang, and P. Moin. Resolution requirements

  9. Rotating flow

    Childs, Peter R N


    Rotating flow is critically important across a wide range of scientific, engineering and product applications, providing design and modeling capability for diverse products such as jet engines, pumps and vacuum cleaners, as well as geophysical flows. Developed over the course of 20 years' research into rotating fluids and associated heat transfer at the University of Sussex Thermo-Fluid Mechanics Research Centre (TFMRC), Rotating Flow is an indispensable reference and resource for all those working within the gas turbine and rotating machinery industries. Traditional fluid and flow dynamics

  10. Of mice and men


    At the end of March , sixty mice were irradiated at the synchro-cyclotron in the course of an experimental programme studying radiation effects on mice and plants (Vicia faba bean roots) being carried out by the CERN Health Physics Group.

  11. A Family of Mice


    @@ 一、故事内容 There is a family of mice in my house. They are father mouse, mother mouse and baby mouse. Baby mouse likes dancing. He is very cute. Father mouse likes watching TV. He likes the sports on TV best. These three mice are clever.

  12. Flow cytometry detection of planktonic cells with polycyclic aromatic hydrocarbons sorbed to cell surfaces

    Cerezo, Maria I.


    Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

  13. Improved and Reproducible Flow Cytometry Methodology for Nuclei Isolation from Single Root Meristem

    Thaís Cristina Ribeiro Silva


    Full Text Available Root meristems have increasingly been target of cell cycle studies by flow cytometric DNA content quantification. Moreover, roots can be an alternative source of nuclear suspension when leaves become unfeasible and for chromosome analysis and sorting. In the present paper, a protocol for intact nuclei isolation from a single root meristem was developed. This proceeding was based on excision of the meristematic region using a prototypical slide, followed by short enzymatic digestion and mechanical isolation of nuclei during homogenization with a hand mixer. Such parameters were optimized for reaching better results. Satisfactory nuclei amounts were extracted and analyzed by flow cytometry, producing histograms with reduced background noise and CVs between 3.2 and 4.1%. This improved and reproducible technique was shown to be rapid, inexpensive, and simple for nuclear extraction from a single root tip, and can be adapted for other plants and purposes.

  14. Validation and quality control of hematolymphoid neoplasm immunophenotyping by flow cytometry

    Wu Lijuan; Xu Dongsheng


    cytometric immunophenotyping has evolved from two-parameter quantitative measurement of peripheral blood lymphocytes to five-or more parameter qualitative evaluation of bone marrow for hematopathology.Leukemia/lymphoma immunophenotyping represent an important addition to histomorphology in the diagnosis,classification and monitoring of hematolymphoid neoplasms. The complexity of five- or more parameter analyses and the interpretation of the data rely on standardization and validation of the instrument,the reagent and the procedure.In addition,clinical flow cytometry laboratories in U.S.are required to document proficiency testing,sample preparation,method accuracy,specificity,sensitivity and precision.CLSI and the U.S.-Canadian Consensus Conference have provided recommendations,but each laboratory is responsible for validating its own qualitative and quantitative procedures.This paper introduces the procedures for quality control of all levels of the operation in a clinical flow cytometry laboratory in USA.

  15. Prognostic relevance of DNA flow cytometry in breast cancer revisited: The 25-year experience of the Portuguese Institute of Oncology of Lisbon

    Pinto, António E.; Pereira, Teresa; Silva, Giovani L.; André, Saudade


    The potential prognostic significance of DNA flow cytometric measurements (DNA ploidy and S-phase fraction) in breast cancer remains in dispute. Inconclusive data, primarily due to the lack of consistent standardization and quality control programs, have limited its translation into clinical practice. The aim of the present review, based on the 25-year experience of the Portuguese Institute of Oncology of Lisbon, is to assess the clinical relevance and application of DNA flow cytometry for the prognosis of breast cancer. Overall, data from Portuguese Institute of Oncology of Lisbon indicate that DNA flow cytometry provides significant prognostic information that is biologically relevant and clinically useful for the management of patients with breast cancer. Furthermore, this data has demonstrated the independent value of DNA aneuploidy as a prognostic indicator of poor clinical outcome in various subgroups of patients with early or locally advanced breast cancer at short- and long-term follow-up. Notably, aneuploidy identifies subsets of patients with grade (G)1 or G2 tumours who exhibit a poor clinical outcome. These patients may benefit from adjuvant chemotherapy, particularly those with luminal A and luminal B/human epidermal growth factor-2-negative endocrine-responsive breast cancer. In conclusion, data from Portuguese Institute of Oncology of Lisbon reinforces the clinical importance and utility of DNA flow cytometric analysis, particularly DNA ploidy, in the prognostic assessment and therapeutic planning for patients with breast cancer. PMID:28454358

  16. Network Flows


    Researchers have suggested other solution strategies, using ideas from nonlinear progamming for solving this general separable convex cost flow problems. Some...plane methods and branch and bound procedures of integer programming, primal-dual methods of linear and nonlinear programming, and polyhedral methods...Combinatorial Optimization: Networks and Matroids), Bazaraa and Jarvis [1978] (Linear Programming and Network Flows), Minieka [1978] (Optimization Algorithms for

  17. Vortical flows

    Wu, Jie-Zhi; Zhou, Ming-De


    This book is a comprehensive and intensive book for graduate students in fluid dynamics as well as scientists, engineers and applied mathematicians. Offering a systematic introduction to the physical theory of vortical flows at graduate level, it considers the theory of vortical flows as a branch of fluid dynamics focusing on shearing process in fluid motion, measured by vorticity. It studies vortical flows according to their natural evolution stages,from being generated to dissipated. As preparation, the first three chapters of the book provide background knowledge for entering vortical flows. The rest of the book deals with vortices and vortical flows, following their natural evolution stages. Of various vortices the primary form is layer-like vortices or shear layers, and secondary but stronger form is axial vortices mainly formed by the rolling up of shear layers.  Problems are given at the end of each chapter and Appendix, some for helping understanding the basic theories, and some involving specific ap...

  18. A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

    Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M


    Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis. CFTR-dependent iodide transport measured by fluorescent quenching of ectopically expressed halide-sensitive yellow fluorescent protein (YFP) is widely being used to study CFTR function by microscopy or plate readers. Since YFP fluorescence in these systems is dependent on YFP expression levels and iodide concentration, differences in sensor expression level between experimental units are normalized at the start of each experiment. To allow accurate measurement of CFTR function by flow cytometry, we reasoned that co-expression of an iodide insensitive fluorescent protein would allow for normalization of sensor expression levels and more accurate quantification of CFTR function. Our data indicated that dsRed and mKate fluorescence are iodide insensitive, and we determined an optimal format for co-expression of these fluorescent proteins with halide-sensitive YFP. We showed using microscopy that ratiometric measurement (YFP/mKate) corrects for differences in sensor expression levels. Ratiometric measurements were essential to accurately measure CFTR function by flow cytometry that we here describe for the first time. Mixing of wild type or mutant CFTR expressing cells indicated that addition of approximately 10% of wild type CFTR expressing cells could be distinguished by ratiometric YFP quenching. Flow cytometric ratiometric YFP quenching also allowed us to study CFTR mutants associated with differential residual function upon ectopic expression. Compared with conventional plate-bound CFTR function assays, the flow cytometric approach described here can be used to study CFTR function in suspension cells. It may be further adapted to study CFTR function in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function by cell sorting.

  19. Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes.

    Webb, Craig; Bedwell, Cathy; Guth, Amanda; Avery, Paul; Dow, Steven


    Oxidative stress and abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, current assays for the reduced form of glutathione (GSH) are time-consuming and semi-quantitative and do not allow assessment of GSH concentrations in individual cell populations. Therefore, we developed a flow cytometric assay for rapid determination of intracellular GSH concentrations in feline blood leukocytes. The assay was based on the ability of the non-fluorescent substrate monochlorobimane (mBCl) to form fluorescent adducts with GSH in a reaction catalyzed by the enzyme glutathione-S-transferase. Using flow cytometry, we found that mBCl was sensitive and specific for intracellular detection of the reduced form of GSH in feline leukocytes. Intracellular GSH concentrations were also stable for at least 24h in EDTA preserved whole blood samples stored at 4 degrees C. Neutrophils and monocytes from normal cats had significantly higher intracellular concentrations of GSH than T cells and B cells. The effects of FIV infection on intracellular GSH concentrations in cats were assessed using flow cytometry. We found that neutrophils from FIV-infected cats had significantly increased GSH concentrations, whereas intracellular GSH concentrations were significantly decreased in CD4(+) and CD8(+) lymphocytes from FIV-infected cats, compared to age-matched control animals. We conclude that a flow cytometric assay based on mBCl may be used to accurately and rapidly assess the effects of various disease states and treatments on GSH concentration in cat leukocytes and to help assess intracellular oxidative stress.

  20. Paradoxical effects of Auger electron-emitting (111)In-DTPA-NLS-CSL360 radioimmunoconjugates on hCD45(+) cells in the bone marrow and spleen of leukemia-engrafted NOD/SCID or NRG mice.

    Bergstrom, Dane; Leyton, Jeffrey V; Zereshkian, Arman; Chan, Conrad; Cai, Zhongli; Reilly, Raymond M


    (111)In-DTPA-NLS-CSL360 radioimmunoconjugates (RIC) recognize the overexpression of the interleukin-3 receptor α-subchain (CD123) relative to the β-subchain (CD131) on leukemia stem cells (LSC). Our aim was to study Auger electron radioimmunotherapy (RIT) of acute myeloid leukemia (AML) with (111)In-DTPA-NLS-CSL360 in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice or NOD-Rag1(null)IL2rγ(null) (NRG) mice engrafted with CD123(+) human AML-5 cells. The toxicity of three doses of (111)In-DTPA-NLS-CSL360 (3.3-4.8MBq; 11-15μg each) injected i.v. every two weeks was studied in non-engrafted NOD/SCID or NRG mice pre-treated with 200cGy of γ-radiation required for AML engraftment. Engraftment efficiency of (1-5)×10(6) cells AML-5 cells inoculated i.v. into NOD/SCID or NRG mice was assessed by flow cytometric analysis for human CD45(+) (hCD45(+)) cells in the bone marrow (BM) and spleen. AML-5 engrafted mice were treated with two or three doses (3.7MBq; 10μg each) every two weeks of (111)In-DTPA-NLS-CSL360, non-specific (111)In-DTPA-NLS-hIgG, unlabeled CSL360 (10μg) or normal saline. The percentage of hCD45(+) cells in the BM and spleen were measured at one week after completion of treatment. (111)In-DTPA-NLS-CSL360 in combination with 200cGy of γ-radiation caused an initial transient decrease in body weight in NOD/SCID but not in NRG mice. There were no hematological, liver or kidney toxicities. The spleen exhibited 13-fold lower engraftment efficiency than the BM in NOD/SCID mice inoculated with 1×10(6) cells but both organs were highly (>85%) engrafted in NRG mice. Unexpectedly, (111)In-DTPA-NLS-CSL360 or non-specific (111)In-DTPA-NLS-hIgG caused a paradoxical 1.5-fold increase (PDTPA-NLS-CSL360 reduced hCD45(+) cells in the spleen by 3.0-fold compared to (111)In-DTPA-NLS-hIgG (P=0.0015) but the proportion of hCD45(+) cells was not significantly different than in normal saline treated mice. Unlabeled CSL360 decreased the percentage of hCD45

  1. Absolute counting of neutrophils in whole blood using flow cytometry.

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K


    Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens.

  2. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    Aanei, Carmen Mariana; Picot, Tiphanie; Tavernier, Emmanuelle; Guyotat, Denis; Campos Catafal, Lydia


    Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoiesis that exhibit heterogeneous clinical presentation and morphological findings, which complicates diagnosis, especially in early stages. Recently, refined definitions and standards in the diagnosis and treatment of MDS were proposed, but numerous questions remain. Multiparameter flow cytometry (MFC) is a helpful tool for the diagnostic workup of patients with suspected MDS, and various scores using MFC data have been developed. However, none of these methods have achieved the sensitivity that is required for a reassuring diagnosis in the absence of morphological abnormalities. One reason may be that each score evaluates one or two lineages without offering a broad view of the dysplastic process. The combination of two scores (e.g., Ogata and Red Score) improved the sensitivity from 50–60 to 88%, but the positive (PPV) and negative predictive values (NPV) must be improved. There are prominent differences between study groups when these scores are tested. Further research is needed to maximize the sensitivity of flow cytometric analysis in MDS. This review focuses on the application of flow cytometry for MDS diagnosis and discusses the advantages and limitations of different approaches. PMID:27446807

  3. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

    Zheng Zhili


    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  4. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  5. L-选择素与小鼠肝癌细胞Hepa1-6淋巴道转移的关系%Correlation between L-selectin and lymphatic metastasis potential of mice hepa1-6 cells

    刘芯宇; 张嘉宁


    目的 研究L-选择素(L-selectin)与其配体甘露糖受体(mannose receptor,MR)间相互作用与小鼠肝癌细胞Hepa1-6淋巴道转移潜能相关性.方法 构建含有L-selectin的重组载体pcDNA3.1,转染小鼠肝癌细胞 Hepa1-6,筛选稳定表达L-selectin的细胞株.采用免疫组化、RT-PCR、流式细胞、体外黏附试验技术研究小鼠肝癌细胞Hepa1-6中L-selectin的表达及其与肿瘤细胞淋巴道转移潜能的相关性.结果 1重组质粒为目的基因L-selectin.2 Hepa1-6细胞表面有L-selectin表达,阳性率20.2%.3流式细胞仪检测结果表明Hepa1-6细胞表达L-selectin为19.9%.4体外黏附试验结果表明,不具有转移特性的Hepa1-6表达L-selectin后可发生淋巴道转移,L-selectin能与MR结合(P<0.001),介导Hepa1-6的淋巴道转移.结论 Hepa1-6表达目的基因L-selectin后具有淋巴道转移潜能,并能与MR在体外结合,介导肿瘤细胞的淋巴道转移.%Objective To study the correlation between L-selectin and its ligand mannose receptor with mice hepatoma cells Hepal-6 lymphatic metastatic potential. Methods The recombinant plasmids pcDNA3. 1 containing L-selectin was constructed, and transfected mouse liver tumor Hepal-6 cells. The cell strains with a stable expression of L-selectin were screened. Immunochemistry,flow cytometric analysis, RT-PCR and other procedures, in addition to in vitro adhesion assays were performed, to verify the expression of L-selectin on the surface of Hepal -6 cells, and the interactions between L-selectin and tumor cells lymphatic metastasis potential was studied. Results The target gene of recombinant plasmid was L-selectin. L-selectin was expressed on Hepal-6 cell' s surface with the positive rate of 20. 2% . By flow cytometric analysis,the positive expression rate of L-selectin in Hepal-6 cells was 19. 9% . In vitro adhesion assays indicated that this protein was able to bind specifically to MR(P < 0.001) ,and further confirmed that Hepal-6 cells

  6. Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand



    It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT)mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times.After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.

  7. Lubrication Flows.

    Papanastasiou, Tasos C.


    Discusses fluid mechanics for undergraduates including the differential Navier-Stokes equations, dimensional analysis and simplified dimensionless numbers, control volume principles, the Reynolds lubrication equation for confined and free surface flows, capillary pressure, and simplified perturbation techniques. Provides a vertical dip coating…

  8. Flow virometric sorting and analysis of HIV quasispecies from plasma

    Jones, Jennifer C.; Keele, Brandon F.; Jenkins, Lisa M. Miller; Demberg, Thorsten


    Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens. PMID:28239654

  9. Sex-sorting sperm using flow cytometry/cell sorting.

    Garner, Duane L; Evans, K Michael; Seidel, George E


    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  10. MICE Particle Identification System

    Bogomilov, M


    The Muon Ionization Cooling Experiment, MICE, at the ISIS accelerator lo- cated at the Rutherford Appleton Laboratory, UK, will be the first experiment to study muon cooling at high precision. Demonstration of muon ionization cooling is an essential step towards the construction of a neutrino factory or a muon collider. Muons are produced by pion decay in a superconducting solenoid and reach MICE with a range of emittances and momenta. The purity of the muon beam is ensured by a system of particle detectors we will briefly describe here.

  11. The use of flow cytometry in assessing malignancy in bone and soft tissue tumors.

    Mankin, Henry J; Fondren, Gertrud; Hornicek, Francis J; Gebhardt, Mark C; Rosenberg, Andrew E


    Since 1982, the orthopaedic research laboratories at the authors' hospital has done flow cytometric and more recently cytofluorometric deoxyribonucleic ploidic analyses of samples of bone and soft tissue tumors. The current authors attempt to define the value of such studies in distinguishing benign from malignant tumors, in conforming to stage of the tumors, and in helping to predict metastasis and death. The series consists of 1134 patients in whom the disease was verified and the survival data were available as a result of a questionnaire study. Statistically, the ploidic analyses were of remarkable value in defining malignancy and in correlating with the stage of the lesion. They were of less value in predicting survival, particularly for patients with osteosarcoma and chondrosarcoma, but seemed to predict survival effectively for patients with soft tissue sarcomas.

  12. Dynamics of the induced acrosome reaction in boar sperm evaluated by flow cytometry

    Birck, Anders; Labouriau, Rodrigo; Christensen, Preben


    The present study investigated the dynamics of the in vitro induced acrosome reaction (AR) in boar sperm in response to medium composition, incubation time and ionophore concentration. The AR is a prerequisite for normal sperm fertilizing capability and can be studied in vitro following induction...... induced AR. A detailed description of the dynamics of sperm viability and acrosomal status of boar sperm following in vitro induction of the AR has to our knowledge not previously been conducted. In the present study, a triple color flow cytometric detection technique was used, which gave simultaneous...... information on sperm viability and acrosomal status. The ionophore induced AR was dependent on extracellular Ca2+, but could be easily induced in boar sperm without capacitation. Capacitation-associated plasma membrane phospholipid scrambling was assessed and a medium specific ability to induce these membrane...

  13. Monitoring of Legionella pneumophila viability after chlorine dioxide treatment using flow cytometry.

    Mustapha, Pascale; Epalle, Thibaut; Allegra, Séverine; Girardot, Françoise; Garraud, Olivier; Riffard, Serge


    The viability of three Legionella pneumophila strains was monitored after chlorine dioxide (ClO2) treatment using a flow cytometric assay. Suspensions of L. pneumophila cells were submitted to increasing concentrations of ClO2. Culturable cells were still detected when using 4 mg/L, but c