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Sample records for platelets flow cytometry

  1. Investigation of platelet function and platelet disorders using flow cytometry.

    Science.gov (United States)

    Rubak, Peter; Nissen, Peter H; Kristensen, Steen D; Hvas, Anne-Mette

    2016-01-01

    Patients with thrombocytopenia or platelet disorders are at risk of severe bleeding. We report the development and validation of flow cytometry assays to diagnose platelet disorders and to assess platelet function independently of platelet count. The assays were developed to measure glycoprotein levels (panel 1) and platelet function (panel 2) in sodium citrated blood. Twenty healthy volunteers and five patients diagnosed with different platelet disorders were included. Glycoprotein expression levels of the receptors Ia, Ib, IIb, IIIa and IX were measured and normalised with forward scatter (FS) as a measurement of platelet size. Platelet function was assessed by CD63, P-selectin and bound fibrinogen in response to arachidonic acid, adenosine diphosphate (ADP), collagen-related peptide, ristocetin and thrombin receptor-activation peptide-6. All patients except one with suspected δ-granule defect showed aberrant levels of glycoproteins in panel 1. Glanzmann's thrombasthenia and genetically verified Bernard-Soulier syndrome could be diagnosed using panel 1. All patients showed reduced platelet function according to at least one agonist. Using panel 2 it was possible to diagnose Bernard-Soulier syndrome, δ-granule defect and GPVI disorder. By combining the two assays, we were able to diagnose different platelet disorders and investigate platelet function independent of platelet count.

  2. Platelet antibody detection by flow cytometry: an effective method to evaluate and give transfusional support in platelet refractoriness

    Directory of Open Access Journals (Sweden)

    Carolina Bonet Bub

    2013-01-01

    Full Text Available BACKGROUND: Immune platelet refractoriness is mainly caused by human leukocyte antigen antibodies (80-90% of cases and, to a lesser extent, by human platelet antigen antibodies. Refractoriness can be diagnosed by laboratory tests and patients should receive compatible platelet transfusions. A fast, effective and low cost antibody-screening method which detects platelet human leukocyte/platelet antigen antibodies is essential in the management of immune platelet refractoriness. OBJECTIVE: The aim of this study was to evaluate the efficiency of the flow cytometry platelet immunofluorescence test to screen for immune platelet refractoriness. METHODS: A group of prospective hematologic patients with clinically suspected platelet refractoriness treated in a referral center in Campinas, SP during July 2006 and July 2011 was enrolled in this study. Platelet antibodies were screened using the flow cytometry platelet immunofluorescence test. Anti-human leukocyte antigen antibodies were detected by commercially available methods. The sensitivity, specificity and predictive values of the immunofluorescence test were determined taking into account that the majority of antiplatelet antibodies presented human leukocyte antigen specificity. RESULTS: Seventy-six samples from 32 female and 38 male patients with a median age of 43.5 years (range: 5-84 years were analyzed. The sensitivity of the test was 86.11% and specificity 75.00% with a positive predictive value of 75.61% and a negative predictive value of 85.71%. The accuracy of the method was 80.26%. CONCLUSION: This study shows that the flow cytometry platelet immunofluorescence test has a high correlation with the anti-human leukocyte antigen antibodies. Despite a few limitations, the method seems to be efficient, fast and feasible as the initial screening for platelet antibody detection and a useful tool to crossmatch platelets for the transfusional support of patients with immune platelet refractoriness.

  3. Flow cytometry-based platelet function testing is predictive of symptom burden in a cohort of bleeders.

    Science.gov (United States)

    Boknäs, Niklas; Ramström, Sofia; Faxälv, Lars; Lindahl, Tomas L

    2017-09-12

    Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p = 0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.

  4. Bacterial screening of platelet concentrates on day 2 and 3 with flow cytometry: the optimal sampling time point?

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    Vollmer, Tanja; Schottstedt, Volkmar; Bux, Juergen; Walther-Wenke, Gabriele; Knabbe, Cornelius; Dreier, Jens

    2014-07-01

    There is growing concern on the residual risk of bacterial contamination of platelet concentrates in Germany, despite the reduction of the shelf-life of these concentrates and the introduction of bacterial screening. In this study, the applicability of the BactiFlow flow cytometric assay for bacterial screening of platelet concentrates on day 2 or 3 of their shelf-life was assessed in two German blood services. The results were used to evaluate currently implemented or newly discussed screening strategies. Two thousand and ten apheresis platelet concentrates were tested on day 2 or day 3 after donation using BactiFlow flow cytometry. Reactive samples were confirmed by the BacT/Alert culture system. Twenty-four of the 2,100 platelet concentrates tested were reactive in the first test by BactiFlow. Of these 24 platelet concentrates, 12 were false-positive and the other 12 were initially reactive. None of the microbiological cultures of the initially reactive samples was positive. Parallel examination of 1,026 platelet concentrates by culture revealed three positive platelet concentrates with bacteria detected only in the anaerobic culture bottle and identified as Staphylococcus species. Two platelet concentrates were confirmed positive for Staphylcoccus epidermidis by culture. Retrospective analysis of the growth kinetics of the bacteria indicated that the bacterial titres were most likely below the diagnostic sensitivity of the BactiFlow assay (screening of platelet concentrates independently of the testing day and the screening strategy. Although the optimal screening strategy could not be defined, this study provides further data to help achieve this goal.

  5. The value of flow cytometry in the measurement of platelet activation and aggregation in human immunodeficiency virus infection.

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    Nkambule, Bongani B; Davison, Glenda; Ipp, Hayley

    2015-01-01

    Human immunodeficiency deficiency virus (HIV) infection is associated with chronic inflammation and an increased risk of thrombotic events. Activated platelets (PLTs) play an important role in both thrombosis and inflammation, and HIV has been shown to induce PLT activation by both direct and indirect mechanisms. P-selectin (CD62P) is a well-described marker of PLT activation, and PLT glycoprotein (GP) IV (CD36) has been identified as a marker of PLT aggregation. Data on PLT function in the context of HIV infection remain inconclusive. Laboratory techniques, such as flow cytometry, enable the assessment of PLTs in their physiological state and environment, with minimal artifactual in vitro activation and aggregation. In this study, we describe a novel flow cytometry PLT assay, which enabled the measurement of PLT function in HIV infection. Forty-one antiretroviral-naïve HIV-positive individuals and 41 HIV-negative controls were recruited from a clinic in the Western Cape. Platelet function was evaluated by assessing the response of platelets to adenosine diphosphate (ADP) at two concentrations (0.04 mM, 0.2 mM). The percentage expression and mean fluorescence intensity (MFI) of CD62P and CD36 was used to evaluate platelet function. These were then correlated with platelet (PLT) count; CD4 count; % CD38/8; viral load and D-dimers. The % CD62P levels were higher in HIV-positive patients (HIV % CD62P 11.33[5.96-29.36] vs. control 2.48[1.56-6.04]; p infection. We were able to show that, although PLTs are significantly activated in HIV compared to uninfected controls, they retain their functional capacity.

  6. The effect of oxLDL on microvesicle release from platelets, measured by a sensitive flow cytometry method.

    Directory of Open Access Journals (Sweden)

    Tine Bo Nielsen

    2015-11-01

    Full Text Available Microvesicles (MVs are submicron vesicles with sizes of 0.1-1.0-µm in diameter, released from various cell types upon activation or apoptosis. Their involvement in a variety of diseases has been intensively investigated. In blood, platelets are potent MV secretors, and oxLDL, a platelet ligand, induce platelet activation and thus potentially MV secretion. This interaction occurs through binding of oxLDL with CD36, located on the platelet membrane. In this study we investigated the effect of in vitro incubation of platelets with oxLDL on MV release. Furthermore, we compared the results obtained when separating MVs larger than 0.5-µm as a measure of results obtained from less sensitive conventional flow cytometers with MVs below the 0.5-µm limit. MV size-distribution was analysed in plasma from 11 healthy volunteers (4 females, 7 males. MVs were identified as < 1-μm and positive for lactadherin binding and cell specific markers. Platelet rich plasma (PRP was incubated without and with oxLDL or LDL (as control to investigate the impact on platelet activation, evident by release of MVs. Size-calibrated fluorescent beads were used to establish the MV gate, and separate small- and large-size vesicles. CD41+ and CD41+CD36+ MVs increased by 6-8 fold in PRP, when left at room temperature, and the presence of cell specific markers increased. Total MV count was unaffected. Incubations with oxLDL did not increase the MV release or affect the distribution of small- and large-size MVs. We found a large inter-individual variation in the fraction of small- and large-size MVs of 73%. In conclusion, we propose that pro-coagulant activity and activation of platelets induced by interaction of platelet CD36 with oxLDL may not involve release of MVs. Furthermore, our results demonstrate great inter-individual variability in size-distribution of platelet derived MVs and thereby stresses the importance for generation of standardized protocols for MV quantification

  7. Platelet antibody screening by flow cytometry is more sensitive than solid phase red cell adherence assay and lymphocytotoxicity technique: a comparative study in Thai patients.

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    Buakaew, Jarin; Promwong, Charuporn

    2010-01-01

    The objective of this study was to compare the sensitivity and specificity of lymphocytotoxicity test (LCT), solid phase red cell adherence assay (SPRCA) and flow cytometry in detecting platelet reactive antibodies against human leukocyte antigens (HLA) class I and human platelet antigens (HPA). Sera from 38 thrombocytopenic patients and 5 mothers of thrombocytopenic newborns were screened for platelet reactive antibodies by these three methods using screening platelets and/or lymphocytes panels derived from six subjects. The sensitivity and specificity of each method and levels of agreement were analysed. HLA antibodies were found in 18, 17 and 19 out of 43 patients' sera tested by LCT, SPRCA and flow cytometry, respectively. Four out of 43 patients' sera were reactive against HPA by flow cytometry, but were reactive to only 2 sera by SPRCA. Using flow cytometry as the reference method, the sensitivities/specificities of SPRCA and LCT in HLA antibody detection were 84.21/95.83% and 94.73/100%, respectively, with a good strength of agreement. SPRCA had 50% sensitivity and 100% specificity in HPA antibody detection compare to flow cytometry. Flow cytometry appeared to be the most sensitive technique compared with SPRCA and LCT for both HPA and HLA antibody screening. SPRCA sensitivity was too low for HPA antibody detection, but this might be because of the small number of samples. There was one serum from the mother of a baby suffering neonatal alloimmune thrombocytopenia (NAIT), in whom SPRCA could not detect HPA antibodies, while flow cytometry came out positive. Therefore, SPRCA should not be used in NAIT investigation and flow cytometry should be employed instead.

  8. CONGESTIVE HEART FAILURE IN DOGS IS ASSOCIATED WITH INCREASED PLATELET LEUKOCYTE AGGREGATION MEASURED BY FLOW CYTOMETRY

    DEFF Research Database (Denmark)

    Tarnow, Inge; Andreasen, Susanne SH; Olsen, Lisbeth Høier

    2010-01-01

    CONGESTIVE HEART FAILURE IN DOGS IS ASSOCIATED WITH ENHANCED PLATELET-LEUKOCYTE AGGREGATES - A MARKER FOR PLATELET ACTIVATION. I Tarnow1, LH Olsen2, SHS Andreasen2, SG Moesgaard2, CE Rasmussen2, AT Kristensen1, T Falk2. 1Departments of Small Animal Clinical Sciences and 2Animal and Veterinary Basic...... Sciences, Faculty of Life Science, University of Copenhagen, Denmark. Chronic congestive heart failure (CHF) in humans is associated with abnormal hemostasis, and changes in hemostatic biomarkers carry a poor prognosis. CHF in dogs has been associated with plasma markers of hypercoagulability, however...

  9. Whole blood flow cytometry measurements of in vivo platelet activation in critically-Ill patients are influenced by variability in blood sampling techniques.

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    Rondina, Matthew T; Grissom, Colin K; Men, Shaohua; Harris, Estelle S; Schwertz, Hansjorg; Zimmerman, Guy A; Weyrich, Andrew S

    2012-06-01

    Flow cytometry is often used to measure in vivo platelet activation in critically-ill patients. Variability in blood sampling techniques, which may confound these measurements, remains poorly characterized. Platelet activation was measured by flow cytometry performed on arterial and venous blood from 116 critically-ill patients. We determined how variability in vascular sampling site, processing times, and platelet counts influenced levels of platelet-monocyte aggregates (PMA), PAC-1 binding (for glycoprotein (GP) IIbIIIa), and P-selectin (P-SEL) expression. Levels of PMA, but not PAC-1 binding or P-SEL expression, were significantly affected by variability in vascular sampling site. Average PMA levels were approximately 60% higher in whole blood drawn from an arterial vessel compared to venous blood (16.2±1.8% vs. 10.7±1.2%, psampling site, processing times, and platelet counts influence levels of PMA, but not PAC-1 binding or P-SEL expression. These data demonstrate the need for rigorous adherence to blood sampling protocols, particularly when levels of PMA, which are most sensitive to variations in blood collection, are measured for detection of in vivo platelet activation. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Flow Cytometry Section

    Data.gov (United States)

    Federal Laboratory Consortium — The primary goal of the Flow Cytometry Section is to provide the services of state-of-the-art multi-parameter cellular analysis and cell sorting for researchers and...

  11. Flow cytometry bioinformatics.

    Directory of Open Access Journals (Sweden)

    Kieran O'Neill

    Full Text Available Flow cytometry bioinformatics is the application of bioinformatics to flow cytometry data, which involves storing, retrieving, organizing, and analyzing flow cytometry data using extensive computational resources and tools. Flow cytometry bioinformatics requires extensive use of and contributes to the development of techniques from computational statistics and machine learning. Flow cytometry and related methods allow the quantification of multiple independent biomarkers on large numbers of single cells. The rapid growth in the multidimensionality and throughput of flow cytometry data, particularly in the 2000s, has led to the creation of a variety of computational analysis methods, data standards, and public databases for the sharing of results. Computational methods exist to assist in the preprocessing of flow cytometry data, identifying cell populations within it, matching those cell populations across samples, and performing diagnosis and discovery using the results of previous steps. For preprocessing, this includes compensating for spectral overlap, transforming data onto scales conducive to visualization and analysis, assessing data for quality, and normalizing data across samples and experiments. For population identification, tools are available to aid traditional manual identification of populations in two-dimensional scatter plots (gating, to use dimensionality reduction to aid gating, and to find populations automatically in higher dimensional space in a variety of ways. It is also possible to characterize data in more comprehensive ways, such as the density-guided binary space partitioning technique known as probability binning, or by combinatorial gating. Finally, diagnosis using flow cytometry data can be aided by supervised learning techniques, and discovery of new cell types of biological importance by high-throughput statistical methods, as part of pipelines incorporating all of the aforementioned methods. Open standards, data

  12. Static platelet adhesion, flow cytometry and serum TXB2 levels for monitoring platelet inhibiting treatment with ASA and clopidogrel in coronary artery disease: a randomised cross-over study

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    Hedbäck Bo

    2009-06-01

    Full Text Available Abstract Background Despite the use of anti-platelet agents such as acetylsalicylic acid (ASA and clopidogrel in coronary heart disease, some patients continue to suffer from atherothrombosis. This has stimulated development of platelet function assays to monitor treatment effects. However, it is still not recommended to change treatment based on results from platelet function assays. This study aimed to evaluate the capacity of a static platelet adhesion assay to detect platelet inhibiting effects of ASA and clopidogrel. The adhesion assay measures several aspects of platelet adhesion simultaneously, which increases the probability of finding conditions sensitive for anti-platelet treatment. Methods With a randomised cross-over design we evaluated the anti-platelet effects of ASA combined with clopidogrel as well as monotherapy with either drug alone in 29 patients with a recent acute coronary syndrome. Also, 29 matched healthy controls were included to evaluate intra-individual variability over time. Platelet function was measured by flow cytometry, serum thromboxane B2 (TXB2-levels and by static platelet adhesion to different protein surfaces. The results were subjected to Principal Component Analysis followed by ANOVA, t-tests and linear regression analysis. Results The majority of platelet adhesion measures were reproducible in controls over time denoting that the assay can monitor platelet activity. Adenosine 5'-diphosphate (ADP-induced platelet adhesion decreased significantly upon treatment with clopidogrel compared to ASA. Flow cytometric measurements showed the same pattern (r2 = 0.49. In opposite, TXB2-levels decreased with ASA compared to clopidogrel. Serum TXB2 and ADP-induced platelet activation could both be regarded as direct measures of the pharmacodynamic effects of ASA and clopidogrel respectively. Indirect pharmacodynamic measures such as adhesion to albumin induced by various soluble activators as well as SFLLRN

  13. [Automated measurement of reticulocyte count by flow cytometry. II: Analysis of the blood containing abnormal erythrocytes or giant platelets].

    Science.gov (United States)

    Oyamatsu, T; Shimizu, N; Takeuchi, K; Yamamoto, M; Kawai, Y; Watanabe, K; Iri, H

    1989-07-01

    We have examined the influence of erythrocytes containing inclusion bodies, nucleated red cells or giant platelets on the measurement of reticulocyte count by automated machine, R-1000. Correlation of the reticulocyte count between automated and conventional method was extremely good in the blood containing red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling . However, correlation was poor when the sample contained the nucleated red cells. Reticulocyte count was decreased in the blood with significant amounts of nucleated red cells. Since nucleated red cells themselves are not counted as reticulocytes in the machine, this was considered to be due to increased young reticulocytes which frequently appeared with nucleated red cells. Both cold agglutinated red cells and giant platelets apparently influenced the reticulocyte count by the R-1000. These results suggest that red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling do not influence the automatic counting of reticulocytes. Although nucleated red cells, cold agglutinated red cells and giant platelets affected the reticulocyte count, the machine shows abnormal flags in most of above cases (except highly agglutinated red cells), so that one can recount reticulocytes by conventional method. We conclude the machine can safely count the reticulocytes even in the blood containing abnormal red cells or platelets.

  14. Two-Photon Flow Cytometry

    Science.gov (United States)

    Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

    2004-01-01

    Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

  15. Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC).

    Science.gov (United States)

    Basabe-Desmonts, L; Ramstrom, S; Meade, G; O'Neill, S; Riaz, A; Lee, L P; Ricco, A J; Kenny, D

    2010-09-21

    IIbβ3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function. A critical function of platelets is to adhere to regions of damage on blood vessel walls; in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix interactions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

  16. Flow cytometry and cell sorting.

    Science.gov (United States)

    Ibrahim, Sherrif F; van den Engh, Ger

    2007-01-01

    Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology.

  17. Teaching Phagocytosis Using Flow Cytometry

    Directory of Open Access Journals (Sweden)

    John Boothby

    2009-12-01

    Full Text Available Investigative microbiology on protists in a basic teaching laboratory environment is limited by student skill level, ease of microbial culture and manipulation, instrumentation, and time. The flow cytometer is gaining use as a mainstream instrument in research and clinical laboratories, but has had minimal application in teaching laboratories. Although the cost of a flow cytometer is currently prohibitive for many microbiology teaching environments and the number of trained instructors and teaching materials is limited, in many ways the flow cytometer is an ideal instrument for teaching basic microbiology. We report here on a laboratory module to study phagocytosis in Tetrahymena sp. using flow cytometry in a basic microbiology teaching laboratory. Students and instructors found the flow cytometry data analysis program, Paint-A-GatePRO-TM, to be very intuitive and easy to learn within a short period of time. Assessment of student learning about Tetrahymena sp., phagocytosis, flow cytometry, and investigative microbiology using an inquiry-based format demonstrated an overall positive response from students.

  18. Flow cytometry of murine spermatocytes.

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    Gaysinskaya, Valeriya; Bortvin, Alex

    2015-04-01

    Protocols for purification of murine male germ cells by FACS based on Hoechst 33342 (Ho342) dye staining have been reported and optimized. However, the protocols are often challenging to follow, partly due to difficulties related to sample preparation, instrument parameters, data display, and selection strategies. In addition, troubleshooting of flow cytometry experiments usually requires some fluency in technical principles and instrument specifications and settings. This unit describes setup and procedures for analysis and sorting of male meiotic prophase I (MPI) cells and other germ cells. Included are procedures that guide data acquisition, display, gating, and back-gating critical for optimal data visualization and cell sorting. Additionally, a flow cytometry analysis of spermatogenesis-defective testis is provided to illustrate the applicability of the technique to the characterization and purification of cells from mutant testis.

  19. Single-platelet nanomechanics measured by high-throughput cytometry

    Science.gov (United States)

    Myers, David R.; Qiu, Yongzhi; Fay, Meredith E.; Tennenbaum, Michael; Chester, Daniel; Cuadrado, Jonas; Sakurai, Yumiko; Baek, Jong; Tran, Reginald; Ciciliano, Jordan C.; Ahn, Byungwook; Mannino, Robert G.; Bunting, Silvia T.; Bennett, Carolyn; Briones, Michael; Fernandez-Nieves, Alberto; Smith, Michael L.; Brown, Ashley C.; Sulchek, Todd; Lam, Wilbur A.

    2016-10-01

    Haemostasis occurs at sites of vascular injury, where flowing blood forms a clot, a dynamic and heterogeneous fibrin-based biomaterial. Paramount in the clot's capability to stem haemorrhage are its changing mechanical properties, the major drivers of which are the contractile forces exerted by platelets against the fibrin scaffold. However, how platelets transduce microenvironmental cues to mediate contraction and alter clot mechanics is unknown. This is clinically relevant, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders. Here, we report a high-throughput hydrogel-based platelet-contraction cytometer that quantifies single-platelet contraction forces in different clot microenvironments. We also show that platelets, via the Rho/ROCK pathway, synergistically couple mechanical and biochemical inputs to mediate contraction. Moreover, highly contractile platelet subpopulations present in healthy controls are conspicuously absent in a subset of patients with undiagnosed bleeding disorders, and therefore may function as a clinical diagnostic biophysical biomarker.

  20. Flow cytometry: retrospective, fundamentals and recent instrumentation.

    Science.gov (United States)

    Picot, Julien; Guerin, Coralie L; Le Van Kim, Caroline; Boulanger, Chantal M

    2012-03-01

    Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to bring flow cytometry to current maturity. The aim of this paper is to present a complete retrospective of the technique and remind flow cytometry fundamentals before focusing on recent commercial instrumentation.

  1. Flow cytometry: basic principles and applications.

    Science.gov (United States)

    Adan, Aysun; Alizada, Günel; Kiraz, Yağmur; Baran, Yusuf; Nalbant, Ayten

    2017-03-01

    Flow cytometry is a sophisticated instrument measuring multiple physical characteristics of a single cell such as size and granularity simultaneously as the cell flows in suspension through a measuring device. Its working depends on the light scattering features of the cells under investigation, which may be derived from dyes or monoclonal antibodies targeting either extracellular molecules located on the surface or intracellular molecules inside the cell. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This review covers the general principles and selected applications of flow cytometry such as immunophenotyping of peripheral blood cells, analysis of apoptosis and detection of cytokines. Additionally, this report provides a basic understanding of flow cytometry technology essential for all users as well as the methods used to analyze and interpret the data. Moreover, recent progresses in flow cytometry have been discussed in order to give an opinion about the future importance of this technology.

  2. Thrombocytopenia: diagnosis with flow cytometry and antiplatelet antibodies.

    Science.gov (United States)

    Guerra, João Carlos de Campos; Kanayama, Ruth Hissae; Nozawa, Sonia Tsukasa; Ioshida, Márcia Regina; Takiri, Irina Yoko; Lazaro, Robson José; Hamerschlak, Nelson; Rosenfeld, Luiz Gastão Mange; Guerra, Celso Carlos de Campos; Bacal, Nydia Strachman

    2011-06-01

    To identify antiplatelet antibodies by flow cytometry (direct method) in patients with thrombocytopenia. Between January 1997 and March 2004 a total of 15100 patients were referred to the Centro de Hematologia de São Paulo for hematological investigation of several diagnoses (anemia, leukopenia, thrombocytopenia, coagulation abnormalities, adenomegaly, leukemia and others). Of those, 1057 were referred because of thrombocytopenia and were divided into two groups: Group Idiopathic thrombocytopenic purpura, with no identifiable cause; and Group Other thrombocytopenia, which included low normal platelet counts cause to be established, hepatitis C and HIV infection, hypersplenism, EDTA-induced artifacts, laboratory error, and other causes. Flow cytometry immunophenotyping was done in 115 cases to identify platelet autoantibodies (direct method). Of the total number of patients, 1057 (7%) presented low platelet counts, 670 were females (63.4%) and age range of one to 75 years. Of the 115 cases (9.7%) submitted to immunophenotyping, the results were positive in 40% and the test was inconclusive in 5%. Idiopathic thrombocytopenic purpura was found in 52% of patients, more often in women. Hepatitis C virus infection was found in 7% and HIV infection in 1%. Low normal platelet counts were found in 17%, laboratory errors in 6%, and laboratory artifacts in 1% of cases. Platelet autoantibodies were found in 76.9% of all idiopathic thrombocytopenic purpura cases. It was negative in 83.3% of the low normal counts. antiplatelet autoantibodies when present help to diagnose idiopathic thrombocytopenic purpura. When absent, suggest other causes of thrombocytopenia.

  3. Imaging flow cytometry for phytoplankton analysis.

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    Dashkova, Veronika; Malashenkov, Dmitry; Poulton, Nicole; Vorobjev, Ivan; Barteneva, Natasha S

    2017-01-01

    This review highlights the concepts and instrumentation of imaging flow cytometry technology and in particular its use for phytoplankton analysis. Imaging flow cytometry, a hybrid technology combining speed and statistical capabilities of flow cytometry with imaging features of microscopy, is rapidly advancing as a cell imaging platform that overcomes many of the limitations of current techniques and contributed significantly to the advancement of phytoplankton analysis in recent years. This review presents the various instrumentation relevant to the field and currently used for assessment of complex phytoplankton communities' composition and abundance, size structure determination, biovolume estimation, detection of harmful algal bloom species, evaluation of viability and metabolic activity and other applications. Also we present our data on viability and metabolic assessment of Aphanizomenon sp. cyanobacteria using Imagestream X Mark II imaging cytometer. Herein, we highlight the immense potential of imaging flow cytometry for microalgal research, but also discuss limitations and future developments.

  4. Flow cytometry: retrospective, fundamentals and recent instrumentation

    OpenAIRE

    Picot, Julien; Guerin, Coralie L; Le Van Kim, Caroline; Boulanger, Chantal M.

    2012-01-01

    Flow cytometry is a complete technology given to biologists to study cellular populations with high precision. This technology elegantly combines sample dimension, data acquisition speed, precision and measurement multiplicity. Beyond the statistical aspect, flow cytometry offers the possibility to physically separate sub-populations. These performances come from the common endeavor of physicists, biophysicists, biologists and computer engineers, who succeeded, by providing new concepts, to b...

  5. Comparison of VerifyNow-P2Y12 test and Flow Cytometry for monitoring individual platelet response to clopidogrel. What is the cut-off value for identifying patients who are low responders to clopidogrel therapy?

    Directory of Open Access Journals (Sweden)

    Castelli Alfredo

    2009-05-01

    Full Text Available Abstract Background Dual anti-platelet therapy with aspirin and a thienopyridine (DAT is used to prevent stent thrombosis after percutaneous coronary intervention (PCI. Low response to clopidogrel therapy (LR occurs, but laboratory tests have a controversial role in the identification of this condition. Methods We studied LR in patients with stable angina undergoing elective PCI, all on DAT for at least 7 days, by comparing: 1 Flow cytometry (FC to measure platelet membrane expression of P-selectin (CD62P and PAC-1 binding following double stimulation with ADP and collagen type I either in the presence of prostaglandin (PG E1; 2 VerifyNow-P2Y12 test, in which results are reported as absolute P2Y12-Reaction-Units (PRU or % of inhibition (% inhibition. Results Thirty controls and 52 patients were analyzed. The median percentage of platelets exhibiting CD62P expression and PAC-1 binding by FC evaluation after stimulation in the presence of PG E1 was 25.4% (IQR: 21.4–33.1% and 3.5% (1.7–9.4%, respectively. Only 6 patients receiving DAT (11.5% had both values above the 1st quartile of controls, and were defined as LR. Evaluation of the same patients with the VerifyNow-P2Y12 test revealed that the area under the receiver-operating-characteristic (ROC curve was 0.94 (95% CI: 0.84–0.98, p 213 PRU gave the maximum accuracy for the detection of patients defined as having LR by FC. Conclusion In conclusion our findings show that a cut-off value of ≤ 15% inhibition or > 213 PRU in the VerifyNow-P2Y12 test may provide the best accuracy for the identification of patients with LR.

  6. Flow cytometry applications in the food industry.

    Science.gov (United States)

    Comas-Riu, Jaume; Rius, Núria

    2009-08-01

    Flow cytometry has become a valuable tool in food microbiology. By analysing large numbers of cells individually using light-scattering and fluorescence measurements, this technique reveals both cellular characteristics and the levels of cellular components. Flow cytometry has been developed to rapidly enumerate microorganisms; to distinguish between viable, metabolically active and dead cells, which is of great importance in food development and food spoilage; and to detect specific pathogenic microorganisms by conjugating antibodies with fluorochromes, which is of great use in the food industry. In addition, high-speed multiparametric data acquisition, analysis and cell sorting, which allow other characteristics of individual cells to be studied, have increased the interest of food microbiologists in this technique. This mini-review gives an overview of the principles of flow cytometry and examples of the application of this technique in the food industry.

  7. Near infrared lasers in flow cytometry.

    Science.gov (United States)

    Telford, William G

    2015-07-01

    Technology development in flow cytometry has closely tracked laser technology, the light source that flow cytometers almost exclusively use to excite fluorescent probes. The original flow cytometers from the 1970s and 1980s used large water-cooled lasers to produce only one or two laser lines at a time. Modern cytometers can take advantage of the revolution in solid state laser technology to use almost any laser wavelength ranging from the ultraviolet to the near infrared. Commercial cytometers can now be equipped with many small solid state lasers, providing almost any wavelength needed for cellular analysis. Flow cytometers are now equipped to analyze 20 or more fluorescent probes simultaneously, requiring multiple laser wavelengths. Instrument developers are now trying to increase this number by designing fluorescent probes that can be excited by laser wavelength at the "edges" of the visible light range, in the near ultraviolet and near-infrared region. A variety of fluorescent probes have been developed that excite with violet and long wavelength ultraviolet light; however, the near-infrared range (660-800 nm) has yet seen only exploitation in flow cytometry. Fortunately, near-infrared laser diodes and other solid state laser technologies appropriate for flow cytometry have been in existence for some time, and can be readily incorporated into flow cytometers to accelerate fluorescent probe development. The near infrared region represents one of the last "frontiers" to maximize the number of fluorescent probes that can be analyzed by flow cytometry. In addition, near infrared fluorescent probes used in biomedical tracking and imaging could also be employed for flow cytometry with the correct laser wavelengths. This review describes the available technology, including lasers, fluorescent probes and detector technology optimal for near infrared signal detection. Published by Elsevier Inc.

  8. Flow cytometry, fluorescent probes, and flashing bacteria

    NARCIS (Netherlands)

    Bunthof, C.J.

    2002-01-01

     


    Key words: fluorescent probes, flow cytometry, CSLM, viability, survival, microbial physiology, lactic acid bacteria, Lactococcus lactis , Lactobacillus plantarum , cheese, milk, probiotic In food industry there is a perceived need for rapid methods for detection and viability a

  9. Supercontinuum white light lasers for flow cytometry

    Science.gov (United States)

    Telford, William G.; Subach, Fedor V.; Verkhusha, Vladislav V.

    2009-01-01

    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

  10. Chromosome analysis and sorting using flow cytometry.

    Science.gov (United States)

    Doležel, Jaroslav; Kubaláková, Marie; Cíhalíková, Jarmila; Suchánková, Pavla; Simková, Hana

    2011-01-01

    Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.

  11. Flow cytometry in oceanography: Status and prospects

    Science.gov (United States)

    Chisholm, Sallie W.; Olson, Robert J.; Yentsch, Clarice M.

    The technology of flow cytometry and cell sorting has existed for ˜20 years, and its potential applications to oceanography have been obvious to many for nearly as long. Its introduction into oceanography did not occur, however, until the early 1980s [Yentsch et al. 1983; Olson et al. 1983]. The introduction was made possible largely t hrough the funding of instruments dedicated to oceanographic applications by the U.S. National Science Foundation, the Office of Naval Research, and private institutions. In the last 5 years, interest in this new tool has grown significantly, and the number of flow cytometry facilities dedicated to applications in oceanography and limnology in the United States, Canada, and Europe has expanded to a surprising degree (Table 1).

  12. Accreditation of flow cytometry in Europe.

    Science.gov (United States)

    Sack, Ulrich; Barnett, David; Demirel, Gulderen Yanikkaya; Fossat, Chantal; Fricke, Stephan; Kafassi, Nikolitsa; Nebe, Thomas; Psarra, Katherina; Steinmann, Jörg; Lambert, Claude

    2013-05-01

    ISO 15189 has been introduced to enable any clinical laboratory, irrespective of geographic location, to be accredited against internationally recognized standards and therefore facilitate direct international comparison of laboratories. Together with increasing use of ISO 15189 for standardization and competition purposes, often triggered by demands of patients and clinicians, clinical flow cytometry laboratories are becoming increasingly challenged to introduce compliant quality management systems. Whilst in most countries, ISO 15189 accreditation is not yet compulsory, there is increasing evidence to suggest that the implementation of this standard is growing. As a result, the European Society of Clinical Cell Analysis (ESCCA) has analysed the impact of accreditation in clinical flow cytometry laboratories. It found, through a discussion forum, that staff qualification, adaptation of multicolour antibody panels, and implementation of a comprehensive quality system (including quality assessment) have been identified as major challenges.

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

    Directory of Open Access Journals (Sweden)

    Kepler Thomas B

    2008-06-01

    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.

  14. Applications of Imaging Flow Cytometry for Microalgae.

    Science.gov (United States)

    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

    2016-01-01

    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.

  15. Characterization of Platelet-Associated Immunoglobulin by Flow Cytometry*%血小板相关免疫球蛋白在流式细胞仪检测上的特征

    Institute of Scientific and Technical Information of China (English)

    王兆钺

    2001-01-01

    血小板相关IgG(PAIgG)常用于ITP与其它免疫性血小板减少的诊断.本研究用流式细胞仪(FCM)检测了47例ITP(包括Evans综合症)、13例非免疫性血小板减少、10例无血小板减少的自身免疫性溶血性贫血(AIHA)和31例正常人的PAIgG,并与ELISA测定的结果进行比较.FCM检测的ITP患者PAIgG的荧光强度(MFI)(2.26±2.29)明显高于非免疫性血小板减少(0.33±0.39),AIHA(0.17±0.07)和正常对照组(0.25±0.15)(P值均小于0.01).同时,ITP患者PAIgG阳性血小板百分率[(44.1±29.0)%]也明显高于非免疫性血小板减少[(17.5±9.4)%],AIHA[(10.7±7.5)%]和正常对照[(16.6±8.4)%](P值均小于0.01).有13例ITP患者(23.4%)的血小板荧光峰形有异常(双峰或峰拖尾),其中有7例只出现峰形异常而无MFI或阳性血小板百分率的改变.这种情况在正常人的血小板未观察到,这可能反映出不同的血小板群体有诊断价值.用FCM测定ITP患者PAIgG总的阳性率为87.2%,稍高于用ELISA检测的83.0%阳性率,但两者之间无显著性差异.两种检测结果的符合率为85.1%.本研究表明,FCM是一个快速与敏感的测定PAIgG的方法,可成为检测免疫性血小板减少的常规的新方法.%Measurement of platelet -associated imunoglobulin (Palg)has frequently been applied for the diagnosis of idiopathic thrombocytopenic purpura(ITP)and other immune thrombocytopenias .In the present study, a flow cytometry (FCM)analysis has been used to detect and characterize Palg in 47 patients with ITP and Evans'syndrome,13patients with non-immune thrombocytopenia,10 patients with autoimmune hemolytic anemia (AIHA) whose pltelet counts were in normal range, and 31 healthy volunteers. Withs FCM measurement, mean fluorescence intensity(MFI)of platelets from patients with ITP and Evans'syndrome (2.26±2.29)was significantly higher than those from non immune thrombocytopenia (0.33±0.39),AIHA(0.17±0.07) and control subjects (0.25±0.15)(P<0.01).Meanwhile

  16. Multiparameter Flow Cytometry For Clinical Applications

    Science.gov (United States)

    Stewart, Carleton C.

    1989-06-01

    Flow Cytometry facilities are well established and provide immunophenotyping and DNA content measurement services. The application of immunophenotyping has been primarily in monitoring therapy and in providing further information to aid in the definitive diagnosis of immunological and neoplastic disease such as: immunodeficiency disease, auto immune disease, organ transplantation, and leukemia and lymphoma. DNA content measurements have been particularly important in determining the fraction of cycling cells and presence of aneuploid cells in neoplasia. This information has been useful in the management of patients with solid tumors.

  17. Honey bee hemocyte profiling by flow cytometry.

    Science.gov (United States)

    Marringa, William J; Krueger, Michael J; Burritt, Nancy L; Burritt, James B

    2014-01-01

    Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

  18. Mixture modeling approach to flow cytometry data.

    Science.gov (United States)

    Boedigheimer, Michael J; Ferbas, John

    2008-05-01

    Flow Cytometry has become a mainstay technique for measuring fluorescent and physical attributes of single cells in a suspended mixture. These data are reduced during analysis using a manual or semiautomated process of gating. Despite the need to gate data for traditional analyses, it is well recognized that analyst-to-analyst variability can impact the dataset. Moreover, cells of interest can be inadvertently excluded from the gate, and relationships between collected variables may go unappreciated because they were not included in the original analysis plan. A multivariate non-gating technique was developed and implemented that accomplished the same goal as traditional gating while eliminating many weaknesses. The procedure was validated against traditional gating for analysis of circulating B cells in normal donors (n = 20) and persons with Systemic Lupus Erythematosus (n = 42). The method recapitulated relationships in the dataset while providing for an automated and objective assessment of the data. Flow cytometry analyses are amenable to automated analytical techniques that are not predicated on discrete operator-generated gates. Such alternative approaches can remove subjectivity in data analysis, improve efficiency and may ultimately enable construction of large bioinformatics data systems for more sophisticated approaches to hypothesis testing.

  19. Microfluidics in flow cytometry and related techniques.

    Science.gov (United States)

    Béné, M C

    2017-05-01

    Technological advances in laboratory automation are now well understood and applied as they considerably improved the speed and robustness of haematological laboratory data, in the companion fields of blood analyzers and flow cytometry. Still rather confidential is the field of microfluidics, mostly confined so far to academic settings and research laboratories. The literature in the field of microfluidics is growing and applications in hematology range from cell counting to flow cytometry, cell sorting, or ex vivo testing. A literature search allows to identify many innovative solutions developed to master the specific physics of fluid movements in microchips. Miniaturization also dwells on findings that have emerged from different areas such as electronics and nanoengineering. This review proposes an overview of the major principles guiding developments in microfluidics and describes a necessarily limited and nonexhaustive series of specific applications. Readers are strongly encouraged to consult the documents referred to in the references section to learn more about this world knocking at our door and possibly liable to revolutionize our profession of hematology biologists in a not so far future. © 2017 John Wiley & Sons Ltd.

  20. Application of flow cytometry to wine microorganisms.

    Science.gov (United States)

    Longin, Cédric; Petitgonnet, Clément; Guilloux-Benatier, Michèle; Rousseaux, Sandrine; Alexandre, Hervé

    2017-04-01

    Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology.

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

    Science.gov (United States)

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

    1996-12-01

    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.

  2. Assessment of Equine Autoimmune Thrombocytopenia (EAT by flow cytometry

    Directory of Open Access Journals (Sweden)

    Schwarzwald Colin

    2001-04-01

    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.

  3. Dynamic proliferation assessment in flow cytometry.

    Science.gov (United States)

    Diermeier-Daucher, Simone; Brockhoff, Gero

    2010-09-01

    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.

  4. Immunological techniques: ELISA, flow cytometry, and immunohistochemistry.

    Science.gov (United States)

    Ford, Pauline J

    2010-01-01

    Techniques to analyze the host immune response elicited by the presence of oral microorganisms and their products are central to our understanding of the local and systemic effects of oral diseases. This immune response has been extensively investigated for periodontal disease. The local response may result in lesions involving the gingival tissues and depending upon host susceptibility and microbial virulence may lead to local tissue destruction. More recently, however, the importance of the systemic inflammatory and immune response to oral organisms has been recognized. These systemic responses have been associated with an increased risk for cardiovascular disease, diabetes, and preterm low birth weight. A number of techniques are used extensively by researchers investigating humoral and cellular immune responses to oral organisms both in local oral tissues and fluids and systemically in peripheral blood. These are enzyme-linked immunosorbent assay (ELISA) to quantify specific antibody and cytokines in serum, gingival crevicular fluid (GCF), and saliva; characterization of T cells from peripheral blood and gingival tissues using flow cytometry; and immunohistological analysis of the inflammatory cell infiltrate in gingival tissues.

  5. A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

    Directory of Open Access Journals (Sweden)

    Alice Assinger

    Full Text Available Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar(+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

  6. Standardization of cytokine flow cytometry assays

    Directory of Open Access Journals (Sweden)

    Cox Josephine

    2005-06-01

    Full Text Available Abstract Background Cytokine flow cytometry (CFC or intracellular cytokine staining (ICS can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online. Results Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC were shipped to various sites, where ICS assays using cytomegalovirus (CMV pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V. ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24% for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82% for samples with a mean of Conclusion ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more

  7. An active, collaborative approach to learning skills in flow cytometry.

    Science.gov (United States)

    Fuller, Kathryn; Linden, Matthew D; Lee-Pullen, Tracey; Fragall, Clayton; Erber, Wendy N; Röhrig, Kimberley J

    2016-06-01

    Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula. Copyright © 2016 The American Physiological Society.

  8. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

    National Research Council Canada - National Science Library

    Heidmann, Iris; Schade-Kampmann, Grit; Lambalk, Joep; Ottiger, Marcel; Di Berardino, Marco

    2016-01-01

    .... Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade...

  9. A model for harmonizing flow cytometry in clinical trials.

    Science.gov (United States)

    Maecker, Holden T; McCoy, J Philip; Amos, Michael; Elliott, John; Gaigalas, Adolfas; Wang, Lili; Aranda, Richard; Banchereau, Jacques; Boshoff, Chris; Braun, Jonathan; Korin, Yael; Reed, Elaine; Cho, Judy; Hafler, David; Davis, Mark; Fathman, C Garrison; Robinson, William; Denny, Thomas; Weinhold, Kent; Desai, Bela; Diamond, Betty; Gregersen, Peter; Di Meglio, Paola; DiMeglio, Paola; Nestle, Frank O; Nestle, Frank; Peakman, Mark; Villanova, Federica; Villnova, Federica; Ferbas, John; Field, Elizabeth; Kantor, Aaron; Kawabata, Thomas; Komocsar, Wendy; Lotze, Michael; Nepom, Jerry; Ochs, Hans; O'Lone, Raegan; Phippard, Deborah; Plevy, Scott; Rich, Stephen; Roederer, Mario; Rotrosen, Dan; Yeh, Jung-Hua

    2010-11-01

    Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

  10. Computational analysis of high-throughput flow cytometry data

    Science.gov (United States)

    Robinson, J Paul; Rajwa, Bartek; Patsekin, Valery; Davisson, Vincent Jo

    2015-01-01

    Introduction Flow cytometry has been around for over 40 years, but only recently has the opportunity arisen to move into the high-throughput domain. The technology is now available and is highly competitive with imaging tools under the right conditions. Flow cytometry has, however, been a technology that has focused on its unique ability to study single cells and appropriate analytical tools are readily available to handle this traditional role of the technology. Areas covered Expansion of flow cytometry to a high-throughput (HT) and high-content technology requires both advances in hardware and analytical tools. The historical perspective of flow cytometry operation as well as how the field has changed and what the key changes have been discussed. The authors provide a background and compelling arguments for moving toward HT flow, where there are many innovative opportunities. With alternative approaches now available for flow cytometry, there will be a considerable number of new applications. These opportunities show strong capability for drug screening and functional studies with cells in suspension. Expert opinion There is no doubt that HT flow is a rich technology awaiting acceptance by the pharmaceutical community. It can provide a powerful phenotypic analytical toolset that has the capacity to change many current approaches to HT screening. The previous restrictions on the technology, based on its reduced capacity for sample throughput, are no longer a major issue. Overcoming this barrier has transformed a mature technology into one that can focus on systems biology questions not previously considered possible. PMID:22708834

  11. 血小板微颗粒的促凝功能与流式细胞术绝对计数值的相关性%Correlation between the coagulation function of the platelet-derived mi-crovesicle and the absolute count of flow cytometry cell sorting

    Institute of Scientific and Technical Information of China (English)

    饶冬东; 张福辉; 薛晓光; 邱君

    2015-01-01

    目的:探讨流式细胞术所获得血小板微颗粒计数与其功能间的关系。方法选取本院2012年9月~2014年9月的100例健康孕产妇及患有合并症孕产妇患者的血液样本作为研究对象,经流式细胞术计数分析及三种功能分析,探讨其相关性。结果流式细胞术获得的乳黏素蛋白促凝血的微粒体计数与Zymuphen MP活性呈弱相关(r越0.5370,P<0.01);与内在凝血酶潜力ETP呈正相关(r越0.7444,P<0.01);与STA磷脂(PPL)促凝分析呈负相关(r=-0.7872,P<0.01)。膜联蛋白V+及促凝血的血小板源性微颗粒的含量水平与功能分析一致。结论血小板微颗粒的促凝功能与流式细胞术绝对计数值密切相关,多参数的使用将会提供更多的生物学信息。%Objective To explore the relationship between the number of platelet-derived microvesicle sorted by flow cytometry cell sorting and their function. Methods 100 copies of blood samplesobtained from healthy maternal women or maternal women with complications from September 2012 to September 2014 in our hospital were selected as the re-search object.The number of platelet microvesicles were calculated by flow cytometry cell sorting,and their functions were recorded by three function analysis.Their relationship was explored. Results The number of platelet microvesicles was slightly correlated with Zymuphen MP activity (r=0.5370,P<0.01) and positively correlated with ETP (r=0.7444,P<0.01),while negatively correlated with STA PPL (r=-0.7872,P<0.01).Membrane associated protein V+was related to the number of coagulation of platelet microvesicles,which was helpful for function analysis. Conclusion The coagulation of platelet microvesicles is closely related to their number counted by flow cytometry cell sorting and the application of multiple parameters will provide valuable biological information.

  12. Flow cytometry evaluation of cell-mediated cytotoxicity.

    Science.gov (United States)

    Zarcone, D; Tilden, A B; Cloud, G; Friedman, H M; Landay, A; Grossi, C E

    1986-11-20

    A novel flow cytometry method for the evaluation of cell-mediated cytotoxicity is described. This method uses flow cytometry analysis to distinguish target cells from effector cells by differences in volume and light scatter characteristics. Non-viable target cells, following their interaction with effector cells, are determined via propidium iodide (PI) dye exclusion and then expressed as a percentage of the total target cell population. This assay is suitable both for analysis of systems which allow recycling of cytotoxic effector cells (total cell cytotoxicity assays, TCCA), and of systems in which recycling does not occur (single cell cytotoxicity assays, SCCA). Natural killer (NK) cell-mediated cytotoxicity evaluated by flow cytometry is significantly correlated with the standard 51Cr release assay. Flow cytometry can also be used to evaluate the competitive inhibition that certain cell types exert on the cell-mediated killing of NK-sensitive targets. A prerequisite for this assay is that competitor cells and target cells are distinguishable through their volume and light scatter characteristics. Advantages and pitfalls of the flow cytometry method are discussed, in comparison with the 51Cr-release assay.

  13. International Society for Advancement of Cytometry (ISAC) flow cytometry shared resource laboratory (SRL) best practices.

    Science.gov (United States)

    Barsky, Lora W; Black, Michele; Cochran, Matthew; Daniel, Benjamin J; Davies, Derek; DeLay, Monica; Gardner, Rui; Gregory, Michael; Kunkel, Desiree; Lannigan, Joanne; Marvin, James; Salomon, Robert; Torres, Carina; Walker, Rachael

    2016-11-01

    The purpose of this document is to define minimal standards for a flow cytometry shared resource laboratory (SRL) and provide guidance for best practices in several important areas. This effort is driven by the desire of International Society for the Advancement of Cytometry (ISAC) members in SRLs to define and maintain standards of excellence in flow cytometry, and act as a repository for key elements of this information (e.g. example SOPs/training material, etc.). These best practices are not intended to define specifically how to implement these recommendations, but rather to establish minimal goals for an SRL to address in order to achieve excellence. It is hoped that once these best practices are established and implemented they will serve as a template from which similar practices can be defined for other types of SRLs. Identification of the need for best practices first occurred through discussions at the CYTO 2013 SRL Forum, with the most important areas for which best practices should be defined identified through several surveys and SRL track workshops as part of CYTO 2014. © 2016 International Society for Advancement of Cytometry.

  14. A Semiconductor Microlaser for Intracavity Flow Cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Akhil, O.; Copeland, G.C.; Dunne, J.L.; Gourley, P.L.; Hendricks, J.K.; McDonald, A.E.

    1999-01-20

    Semiconductor microlasers are attractive components for micro-analysis systems because of their ability to emit coherent intense light from a small aperture. By using a surface-emitting semiconductor geometry, we were able to incorporate fluid flow inside a laser microcavity for the first time. This confers significant advantages for high throughput screening of cells, particulates and fluid analytes in a sensitive microdevice. In this paper we discuss the intracavity microfluidics and present preliminary results with flowing blood and brain cells.

  15. Principles and applications of flow cytometry and cell sorting in companion animal medicine.

    Science.gov (United States)

    Wilkerson, Melinda J

    2012-01-01

    Flow cytometry measures multiple characteristic of single cells using light scatter properties and fluorescence properties of fluorescent probes with specificity to cellular constituents. The use of flow cytometry in the veterinary clinical laboratory has become more routine in veterinary diagnostic laboratories and institutions (http://www.vet.k-state.edu/depts/dmp/service/immunology/index.htm), and reference laboratories. The most common applications in small animal medicine includes quantitation of erythrocytes and leukocytes in automated hematology instruments, detection of antibodies to erythrocytes and platelets in cases of immune-mediated diseases, immunophenotyping of leukocytes and lymphocytes in immunodeficiency syndromes, or leukemias and lymphomas. DNA content analysis to identify aneuploidy or replicating cells in tumor preparations has not gained routine acceptance because of the variability of prognostic results. Other applications including cell sorting and multiplexing using microspheres are potential assays of the future once they become validated and the instrumentation footprint becomes more and more compact, less expensive, and easier to use.

  16. Lymphoid Neoplasia: Correlations Between Morphology and Flow Cytometry.

    Science.gov (United States)

    Rout, Emily D; Avery, Paul R

    2017-01-01

    Cytology is commonly used to diagnose lymphoma and leukemia. Frequently, a diagnosis of lymphoproliferative disease can be obtained via cytology, and some of the common subtypes of canine lymphoma and leukemia can have characteristic cytologic features. Flow cytometry is a critical tool in the objective diagnosis and further characterization of lymphoma and leukemia. Features of the immunophenotype, such as expression of certain cell surface proteins or cell size, can provide important prognostic information. This review describes the cytologic features, flow cytometry immunophenotype, and immunophenotypic prognostic information for 6 major types of canine lymphoma and leukemia.

  17. Standardization, Calibration, and Control in Flow Cytometry.

    Science.gov (United States)

    Wang, Lili; Hoffman, Robert A

    2017-01-05

    Because flow cytometers are designed to measure particle characteristics, particles are the most common materials used to calibrate, control, and standardize the instruments. Definitions and cautions are provided for common terms to alert the reader to critical distinctions in meaning. This unit presents extensive background on particle types and cautions and describes practical aspects of methods to standardize and calibrate instruments. Procedures are provided to characterize performance in terms of optical alignment, fluorescence and light scatter resolution, and sensitivity. Finally, suggestions follow for analyzing particles used for calibration. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  18. The curvHDR method for gating flow cytometry samples

    Directory of Open Access Journals (Sweden)

    Wand Matthew P

    2010-01-01

    Full Text Available Abstract Background High-throughput flow cytometry experiments produce hundreds of large multivariate samples of cellular characteristics. These samples require specialized processing to obtain clinically meaningful measurements. A major component of this processing is a form of cell subsetting known as gating. Manual gating is time-consuming and subjective. Good automatic and semi-automatic gating algorithms are very beneficial to high-throughput flow cytometry. Results We develop a statistical procedure, named curvHDR, for automatic and semi-automatic gating. The method combines the notions of significant high negative curvature regions and highest density regions and has the ability to adapt well to human-perceived gates. The underlying principles apply to dimension of arbitrary size, although we focus on dimensions up to three. Accompanying software, compatible with contemporary flow cytometry infor-matics, is developed. Conclusion The method is seen to adapt well to nuances in the data and, to a reasonable extent, match human perception of useful gates. It offers big savings in human labour when processing high-throughput flow cytometry data whilst retaining a good degree of efficacy.

  19. Coefficient of variation in flow cytometry of phagocytosis.

    Science.gov (United States)

    Fujikawa-Yamamoto, K; Odashima, S

    1987-01-01

    Fluorescence histograms of V79 Chinese hamster lung cells containing phagocytized fluorescent microspheres were measured by flow cytometry. In the fluorescence histograms, the coefficient of variation (CV) of the peak for cells ingesting microspheres was not constant. Rather, it decreased with the number of microspheres ingested by the cells.

  20. Analysis of repetitive DNA in chromosomes by flow cytometry.

    Science.gov (United States)

    Brind'Amour, Julie; Lansdorp, Peter M

    2011-06-01

    We developed a flow cytometry method, chromosome flow fluorescence in situ hybridization (FISH), called CFF, to analyze repetitive DNA in chromosomes using FISH with directly labeled peptide nucleic acid (PNA) probes. We used CFF to measure the abundance of interstitial telomeric sequences in Chinese hamster chromosomes and major satellite sequences in mouse chromosomes. Using CFF we also identified parental homologs of human chromosome 18 with different amounts of repetitive DNA.

  1. Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow.

    Science.gov (United States)

    Whyte, Claire S; Swieringa, Frauke; Mastenbroek, Tom G; Lionikiene, Ausra S; Lancé, Marcus D; van der Meijden, Paola E J; Heemskerk, Johan W M; Mutch, Nicola J

    2015-04-16

    The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding "cap." These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow.

  2. Method of detaching adherent cells for flow cytometry

    KAUST Repository

    Kaur, Mandeep

    2015-12-24

    In one aspect, a method for detaching adherent cells can include adding a cell lifting solution to the media including a sample of adherent cells and incubating the sample of adherent cells with the cell lifting solution. No scraping or pipetting is needed to facilitate cell detachment. The method do not require inactivation of cell lifting solution and no washing of detaching cells is required to remove cell lifting solution. Detached cells can be stained with dye in the presence of cell lifting solution and are further analyzed using flow cytometer. The method has been tested using 6 different cell lines, 4 different assays, two different plate formats (96 and 384 well plates) and two different flow cytometry instruments. The method is simple to perform, less time consuming, with no cell loss and makes high throughput flow cytometry on adherent cells a reality.

  3. Microfluidic Impedance Flow Cytometry Enabling High-Throughput Single-Cell Electrical Property Characterization

    Directory of Open Access Journals (Sweden)

    Jian Chen

    2015-04-01

    Full Text Available This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1 early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2 microfluidic impedance flow cytometry with enhanced sensitivity; (3 microfluidic impedance and optical flow cytometry for single-cell analysis and (4 integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications.

  4. Microfluidic impedance flow cytometry enabling high-throughput single-cell electrical property characterization.

    Science.gov (United States)

    Chen, Jian; Xue, Chengcheng; Zhao, Yang; Chen, Deyong; Wu, Min-Hsien; Wang, Junbo

    2015-04-29

    This article reviews recent developments in microfluidic impedance flow cytometry for high-throughput electrical property characterization of single cells. Four major perspectives of microfluidic impedance flow cytometry for single-cell characterization are included in this review: (1) early developments of microfluidic impedance flow cytometry for single-cell electrical property characterization; (2) microfluidic impedance flow cytometry with enhanced sensitivity; (3) microfluidic impedance and optical flow cytometry for single-cell analysis and (4) integrated point of care system based on microfluidic impedance flow cytometry. We examine the advantages and limitations of each technique and discuss future research opportunities from the perspectives of both technical innovation and clinical applications.

  5. Characterization of ploidy levels in Chrysanthemum L. by flow cytometry

    Institute of Scientific and Technical Information of China (English)

    Yue-ping Ma; Jiang-xue Wei; Zhi-yang Yu; Bing Qin; Si-lan Dai

    2015-01-01

    Analyzing the ploidy levels of plants is important for identifying species, selecting parental lines, identifying the relationships between species, and determining evolutionary patterns. The genus Chrysanthemum is widely distributed throughout the world and exhibits different ploidy levels. We used flow cytometry to analyze the ploidy levels of nine species of Chrysanthemum L. collected from different regions and geographical locations in China. Three diploids from Henan and Wuhan provinces corresponded to Chrysanthe-mum lavandulifolium and two species of C. nankingense, while three tetraploids from various regions corresponded to C. indicum and two species of C. chanetii. Two hexaploids corresponding to C. vestitum were collected at Funiu moun-tain (Henan province), and C. zawadskii was collected at Huangshan mountain (Anhui province). We found that OTTO extraction buffer was suitable for extracting nuclei from most species, apart from C. zawadskii. Flow cytometry proved to be a simple, rapid, and highly accurate method for identifying ploidy levels in Chrysanthemum species.

  6. An efficient method for enumerating oral spirochetes using flow cytometry.

    Science.gov (United States)

    Orth, Rebecca; O'Brien-Simpson, Neil; Dashper, Stuart; Walsh, Katrina; Reynolds, Eric

    2010-02-01

    Spirochetes, such as Treponema denticola, are thin walled, helical, motile bacteria. They are notoriously difficult to enumerate due to their thinness and the difficulties associated with culturing them. Here we have developed a modified oral bacterial growth medium (OBGM) that significantly improves the cultivation of T. denticola compared with a previously published growth medium. Three methods for the enumeration of T. denticola, semi-solid growth medium colony-forming unit (CFU) counts, DNA analysis and flow cytometry, are described and compared. Enumeration of T. denticola using the semi-solid agar method resulted in a positive linear relationship with absorbance of the culture (R(2)=0.9423). However, the semi-solid agar method was found to consistently underestimate (by 50 fold) the T. denticola cell density compared to previously published data. DNA analysis of T. denticola cultures reliably and consistently resulted in a positive linear relationship with absorbance (R(2)=0.9360), giving a calculated cell density of 6.9 x 10(8)cells/mL at an absorbance of 0.2 at 650 nm. Flow cytometry was also found to result in a positive linear relationship with absorbance (R(2)=0.9874), giving a calculated cell density of 6.6 x 10(8)cells/mL at an absorbance of 0.2 at 650 nm. In comparing all of these enumeration methods, the flow cytometry method was found to have distinct advantages, as it is accurate, rapid, and could distinguish between live and dead bacteria. Thus flow cytometry is a recommended means for the rapid and reliable enumeration of viable spirochetes from culture.

  7. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis

    OpenAIRE

    Heidmann, Iris; Schade-Kampmann, Grit; Lambalk, Joep; Ottiger, Marcel; Di Berardino, Marco

    2016-01-01

    Introduction An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a comm...

  8. Flow Cytometry in the Detection of Neonatal Sepsis

    Directory of Open Access Journals (Sweden)

    Volker N. Umlauf

    2013-01-01

    Full Text Available Neonatal sepsis remains a burden problem by showing minimal initial symptoms of subtle character, nonspecific manifestation, and diagnostic pitfalls. The clinical course can be fulminant and fatal if treatment is not commenced promptly. It is therefore crucial to establish early diagnosis and initiate adequate therapy. Besides clinical symptoms, the most reliable laboratory markers in establishing diagnosis is currently the combined measurement of CRP and a cytokine (IL-6 and IL-8. Due to their different kinetics, a diagnostic gap might occur and thus withholding antimicrobial therapy in clinical suspicion of infection is not acceptable. We therefore need parameters which unerringly differentiate between infants in need for antimicrobial therapy and those who are not. Flow cytometry promises to be a useful tool in this field, allowing the determination of different cellular, dissolved, and functional pathophysiological components of sepsis. Despite technical and methodical advances in flow cytometry, its use in clinical routine is still limited. Advantages and disadvantages of promising new parameters in diagnosis of sepsis performed by flow cytometry, particularly CD64, HLA-DR, and apoptosis, are reviewed here. The necessity of tests to be used as an “ideal” parameter is presented.

  9. Ultraviolet 320 nm laser excitation for flow cytometry.

    Science.gov (United States)

    Telford, William; Stickland, Lynn; Koschorreck, Marco

    2017-04-01

    Although multiple lasers and high-dimensional analysis capability are now standard on advanced flow cytometers, ultraviolet (UV) lasers (usually 325-365 nm) remain an uncommon excitation source for cytometry. This is primarily due to their cost, and the small number of applications that require this wavelength. The development of the Brilliant Ultraviolet (BUV fluorochromes, however, has increased the importance of this formerly niche excitation wavelength. Historically, UV excitation was usually provided by water-cooled argon- and krypton-ion lasers. Modern flow cytometers primary rely on diode pumped solid state lasers emitting at 355 nm. While useful for all UV-excited applications, DPSS UV lasers are still large by modern solid state laser standards, and remain very expensive. Smaller and cheaper near UV laser diodes (NUVLDs) emitting at 375 nm make adequate substitutes for 355 nm sources in many situations, but do not work as well with very short wavelength probes like the fluorescent calcium chelator indo-1. In this study, we evaluate a newly available UV 320 nm laser for flow cytometry. While shorter in wavelength that conventional UV lasers, 320 is close to the 325 nm helium-cadmium wavelength used in the past on early benchtop cytometers. A UV 320 nm laser was found to excite almost all Brilliant Ultraviolet dyes to nearly the same level as 355 nm sources. Both 320 nm and 355 nm sources worked equally well for Hoechst and DyeCycle Violet side population analysis of stem cells in mouse hematopoetic tissue. The shorter wavelength UV source also showed excellent excitation of indo-1, a probe that is not compatible with NUVLD 375 nm sources. In summary, a 320 nm laser module made a suitable substitute for conventional 355 nm sources. This laser technology is available in a smaller form factor than current 355 nm units, making it useful for small cytometers with space constraints. © 2017 International Society for Advancement of Cytometry. © 2017 International

  10. Label-free high-throughput imaging flow cytometry

    Science.gov (United States)

    Mahjoubfar, A.; Chen, C.; Niazi, K. R.; Rabizadeh, S.; Jalali, B.

    2014-03-01

    Flow cytometry is an optical method for studying cells based on their individual physical and chemical characteristics. It is widely used in clinical diagnosis, medical research, and biotechnology for analysis of blood cells and other cells in suspension. Conventional flow cytometers aim a laser beam at a stream of cells and measure the elastic scattering of light at forward and side angles. They also perform single-point measurements of fluorescent emissions from labeled cells. However, many reagents used in cell labeling reduce cellular viability or change the behavior of the target cells through the activation of undesired cellular processes or inhibition of normal cellular activity. Therefore, labeled cells are not completely representative of their unaltered form nor are they fully reliable for downstream studies. To remove the requirement of cell labeling in flow cytometry, while still meeting the classification sensitivity and specificity goals, measurement of additional biophysical parameters is essential. Here, we introduce an interferometric imaging flow cytometer based on the world's fastest continuous-time camera. Our system simultaneously measures cellular size, scattering, and protein concentration as supplementary biophysical parameters for label-free cell classification. It exploits the wide bandwidth of ultrafast laser pulses to perform blur-free quantitative phase and intensity imaging at flow speeds as high as 10 meters per second and achieves nanometer-scale optical path length resolution for precise measurements of cellular protein concentration.

  11. Analysis of chromosome damage for biodosimetry using imaging flow cytometry.

    Science.gov (United States)

    Beaton, L A; Ferrarotto, C; Kutzner, B C; McNamee, J P; Bellier, P V; Wilkins, R C

    2013-08-30

    The dicentric chromosome assay (DCA), which involves counting the frequency of dicentric chromosomes in mitotic lymphocytes and converting it to a dose-estimation for ionizing radiation exposure, is considered to be the gold standard for radiation biodosimetry. Furthermore, for emergency response, the DCA has been adapted for triage by simplifying the scoring method [1]. With the development of new technologies such as the imaging flow cytometer, it may now be possible to adapt this microscope-based method to an automated cytometry method. This technology allows the sensitivity of microscopy to be maintained while adding the increased throughput of flow cytometry. A new protocol is being developed to adapt the DCA to the imaging cytometer in order to further increase the rapid determination of a biological dose. Peripheral blood mononuclear cells (PBMC) were isolated from ex vivo irradiated whole blood samples using a density gradient separation method and cultured with PHA and Colcemid. After 48h incubation, the chromosomes were isolated, stained for DNA content with propidium iodide (PI) and labelled with a centromere marker. Stained chromosomes were then analyzed on the ImageStream(×) (EMD-Millipore, Billerica, MA). Preliminary results indicate that individual chromosomes can be identified and mono- and dicentric chromosomes can be differentiated by imaging cytometry. A dose response curve was generated using this technology. The details of the method and the dose response curve are presented and compared to traditional microscope scoring. Imaging cytometry is a new technology which enables the rapid, automated analysis of fluorescently labelled chromosomes. Adapting the dicentric assay to this technology has the potential for high throughput analysis for mass casualty events.

  12. AutoGate: automating analysis of flow cytometry data.

    Science.gov (United States)

    Meehan, Stephen; Walther, Guenther; Moore, Wayne; Orlova, Darya; Meehan, Connor; Parks, David; Ghosn, Eliver; Philips, Megan; Mitsunaga, Erin; Waters, Jeffrey; Kantor, Aaron; Okamura, Ross; Owumi, Solomon; Yang, Yang; Herzenberg, Leonard A; Herzenberg, Leonore A

    2014-05-01

    Nowadays, one can hardly imagine biology and medicine without flow cytometry to measure CD4 T cell counts in HIV, follow bone marrow transplant patients, characterize leukemias, etc. Similarly, without flow cytometry, there would be a bleak future for stem cell deployment, HIV drug development and full characterization of the cells and cell interactions in the immune system. But while flow instruments have improved markedly, the development of automated tools for processing and analyzing flow data has lagged sorely behind. To address this deficit, we have developed automated flow analysis software technology, provisionally named AutoComp and AutoGate. AutoComp acquires sample and reagent labels from users or flow data files, and uses this information to complete the flow data compensation task. AutoGate replaces the manual subsetting capabilities provided by current analysis packages with newly defined statistical algorithms that automatically and accurately detect, display and delineate subsets in well-labeled and well-recognized formats (histograms, contour and dot plots). Users guide analyses by successively specifying axes (flow parameters) for data subset displays and selecting statistically defined subsets to be used for the next analysis round. Ultimately, this process generates analysis "trees" that can be applied to automatically guide analyses for similar samples. The first AutoComp/AutoGate version is currently in the hands of a small group of users at Stanford, Emory and NIH. When this "early adopter" phase is complete, the authors expect to distribute the software free of charge to .edu, .org and .gov users.

  13. Absolute counting of neutrophils in whole blood using flow cytometry.

    Science.gov (United States)

    Brunck, Marion E G; Andersen, Stacey B; Timmins, Nicholas E; Osborne, Geoffrey W; Nielsen, Lars K

    2014-12-01

    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.

  14. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    Science.gov (United States)

    Aanei, Carmen Mariana; Picot, Tiphanie; Tavernier, Emmanuelle; Guyotat, Denis; Campos Catafal, Lydia

    2016-01-01

    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

  15. Cell-cooling in flow cytometry by Peltier elements.

    Science.gov (United States)

    Göttlinger, C; Meyer, K L; Weichel, W; Müller, W; Raftery, B; Radbruch, A

    1986-05-01

    We have built a cooling device for cell suspensions in flow cytometry that makes use of the Peltier effect (Barnard RD, Thermo electricity in Metals and Alloys, Taylor and Francis, London; Siemens-Z 34:383-88, 1963). The prototype described here is used for cooling collection tubes during long-duration cell sorting and is capable of maintaining a temperature of 2-5 degrees C in a cell suspension of up to 3 ml. In general, Peltier element-based cooling is useful for equilibrating the temperature of small volumes of fluids. Furthermore, Peltier element-based cooling devices are easy to build and handle.

  16. Analysis of receptor tyrosine kinase internalization using flow cytometry.

    Science.gov (United States)

    Li, Ning; Hill, Kristen S; Elferink, Lisa A

    2008-01-01

    The internalization of activated receptor tyrosine kinases (RTKs) by endocytosis and their subsequent down regulation in lysosomes plays a critical role in regulating the duration and intensity of downstream signaling events. Uncoupling of the RTK cMet from ligand-induced degradation was recently shown to correlate with sustained receptor signaling and increased cell tumorigenicity, suggesting that the corruption of these endocytic mechanisms could contribute to increased cMet signaling in metastatic cancers. To understand how cMet signaling for normal cell growth is controlled by endocytosis and how these mechanisms are dysregulated in metastatic cancers, we developed flow cytometry-based assays to examine cMet internalization.

  17. Determination of DNA Content of Aquatic Bacteria by Flow Cytometry

    OpenAIRE

    Button, D. K.; Robertson, Betsy R.

    2001-01-01

    The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed fro...

  18. Fluorescence triggering: A general strategy for enumerating and phenotyping extracellular vesicles by flow cytometry.

    Science.gov (United States)

    Arraud, Nicolas; Gounou, Céline; Turpin, Delphine; Brisson, Alain R

    2016-02-01

    Plasma contains cell-derived extracellular vesicles (EVs) which participate in various physiopathological processes and have potential biomedical applications. Despite intense research activity, knowledge on EVs is limited mainly due to the difficulty of isolating and characterizing sub-micrometer particles like EVs. We have recently reported that a simple flow cytometry (FCM) approach based on triggering the detection on a fluorescence signal enabled the detection of 50× more Annexin-A5 binding EVs (Anx5+ EVs) in plasma than the conventional FCM approach based on light scattering triggering. Here, we present the application of the fluorescence triggering approach to the enumeration and phenotyping of EVs from platelet free plasma (PFP), focusing on CD41+ and CD235a+ EVs, as well as their sub-populations which bind or do not bind Anx5. Higher EV concentrations were detected by fluorescence triggering as compared to light scattering triggering, namely 40× for Anx5+ EVs, 75× for CD41+ EVs, and 15× for CD235a+ EVs. We found that about 30% of Anx5+ EVs were of platelet origin while only 3% of them were of erythrocyte origin. In addition, a majority of EVs from platelet and erythrocyte origin do not expose PS, in contrast to the classical theory of EV formation. Furthermore, the same PFP samples were analyzed fresh and after freeze-thawing, showing that freeze-thawing processes induce an increase, of about 35%, in the amount of Anx5+ EVs, while the other EV phenotypes remain unchanged. The method of EV detection and phenotyping by fluorescence triggering is simple, sensitive and reliable. We foresee that its application to EV studies will improve our understanding on the formation mechanisms and functions of EVs in health and disease and help the development of EV-based biomarkers. © 2015 International Society for Advancement of Cytometry.

  19. Detection of Kinase Translocation Using Microfluidic Electroporative Flow Cytometry

    Science.gov (United States)

    Lu, Chang; Wang, Jun; Bao, Ning; Paris, Leela; Wang, Hsiang-Yu; Geahlen, Robert

    2008-03-01

    Translocation of a protein between different subcellular compartments is a common event during signal transduction in living cells. Detection of these events has been largely carried out based on imaging of a low number of cells and subcellular fractionation/Western blotting. These conventional techniques either lack the high throughput desired for probing an entire cell population or provide only the average behaviors of cell populations without information from single cells. Here we demonstrate a new tool, referred to as microfluidic electroporative flow cytometry, to detect the translocation of an EGFP-tagged tyrosine kinase, Syk, to the plasma membrane in B cells at the level of the cell population. We combine electroporation with flow cytometry and observe the release of intracellular kinase out of the cells during electroporation. We found that the release of the kinase was strongly influenced by its subcellular localization. Cells stimulated through the antigen receptor have a fraction of the kinase at the plasma membrane and retain more kinase after electroporation than do cells without stimulation and translocation. This tool will have utility for kinase-related drug discovery and tumor diagnosis and staging.

  20. Detection of Significant Bacteriuria by Automated Urinalysis Using Flow Cytometry

    Science.gov (United States)

    Okada, Hiroshi; Sakai, Yutaka; Miyazaki, Shigenori; Arakawa, Soichi; Hamaguchi, Yukio; Kamidono, Sadao

    2000-01-01

    A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods. PMID:10921941

  1. THE TECHNIQUE OF FLOW CYTOMETRY IN DIAGNOSTIC RESEARCH

    Directory of Open Access Journals (Sweden)

    Arkadiusz Macheta

    2013-09-01

    Full Text Available Flow cytometry is a technology that simultaneously counts and then examines multiple physical and/or chemical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. The properties measured include a particle’s relative size, relative granularity or internal complexity, and relative fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell scatters incident laser light and emits fluorescence. One of the most significant applications is immunophenotyping of cells - the most important tool in diagnosis and monitoring haematological disorders, such as acute and chronic leukemia, lymphoma, monoclonal gammopathy, myelodisplastic and myeloproliferative diseases.

  2. Digital analysis and sorting of fluorescence lifetime by flow cytometry.

    Science.gov (United States)

    Houston, Jessica P; Naivar, Mark A; Freyer, James P

    2010-09-01

    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

  3. Misty Mountain clustering: application to fast unsupervised flow cytometry gating

    Directory of Open Access Journals (Sweden)

    Sealfon Stuart C

    2010-10-01

    Full Text Available Abstract Background There are many important clustering questions in computational biology for which no satisfactory method exists. Automated clustering algorithms, when applied to large, multidimensional datasets, such as flow cytometry data, prove unsatisfactory in terms of speed, problems with local minima or cluster shape bias. Model-based approaches are restricted by the assumptions of the fitting functions. Furthermore, model based clustering requires serial clustering for all cluster numbers within a user defined interval. The final cluster number is then selected by various criteria. These supervised serial clustering methods are time consuming and frequently different criteria result in different optimal cluster numbers. Various unsupervised heuristic approaches that have been developed such as affinity propagation are too expensive to be applied to datasets on the order of 106 points that are often generated by high throughput experiments. Results To circumvent these limitations, we developed a new, unsupervised density contour clustering algorithm, called Misty Mountain, that is based on percolation theory and that efficiently analyzes large data sets. The approach can be envisioned as a progressive top-down removal of clouds covering a data histogram relief map to identify clusters by the appearance of statistically distinct peaks and ridges. This is a parallel clustering method that finds every cluster after analyzing only once the cross sections of the histogram. The overall run time for the composite steps of the algorithm increases linearly by the number of data points. The clustering of 106 data points in 2D data space takes place within about 15 seconds on a standard laptop PC. Comparison of the performance of this algorithm with other state of the art automated flow cytometry gating methods indicate that Misty Mountain provides substantial improvements in both run time and in the accuracy of cluster assignment. Conclusions

  4. Thyroid hormone effect on human mitochondria measured by flow cytometry

    DEFF Research Database (Denmark)

    Kvetny, Jan; Bomholt, Tobias; Pedersen, Palle

    2009-01-01

    BACKGROUND: Mitochondrial function may be impaired in a number of diseases including metabolic syndrome, cardiovascular disease and endocrine disorders. Therefore it is important to be able to measure mitochondrial function in human cells. PURPOSE: The aim of the present study was to evaluate...... a method to measure mitochondrial function in human derived cells, which also would reflect regulation by thyroid hormones. METHODS: The MDA-MB-231 cell line (a human breast cancer cell line) was incubated with bioactive iodothyronines (T(4), 3'-3, 5-T(3), 3, 5-T(2)) 50 nmol/l for 3 h. Mitochondrial......: It was possible to measure mitochondrial membrane potential (MMP) in human derived cells and to examine thyroid hormone effects using flow cytometry. Bioactive iodothyronines increased mitochondrial membrane potential. TRIAC had no effect and L-Carnitine only inhibited T(4) stimulation of membrane potential...

  5. Dynamic quantification of antigen molecules with flow cytometry

    Science.gov (United States)

    Moskalensky, A.E.; Chernyshev, A.V.; Yurkin, M.A.; Nekrasov, V.M.; Polshchitsin, A.A.; Parks, D.R.; Moore, W.A.; Herzenberg, L.A.; Filatenkov, A.; Maltsev, V.P.; Orlova, D.Y.

    2015-01-01

    Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg–Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions. PMID:25687877

  6. Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach : Report from the Dutch Working Party on Flow Cytometry in MDS

    NARCIS (Netherlands)

    Westers, Theresia M.; van der Velden, Vincent H. J.; Alhan, Canan; Bekkema, Roelof; Bijkerk, Andre; Brooimans, Rik A.; Cali, Claudia; Drager, Angelika M.; de Haas, Valerie; Homburg, Christa; Kuiper-Kramer, P. (Ellen) A.; Leenders, Marije; Lommerse, Ingrid; Marvelde, Jeroen G. te; van der Molen-Sinke, Joke K.; Moshaver, Bijan; Mulder, Andre B.; Preijers, Frank W. M. B.; Schindhelm, Roger K.; van der Sluijs, Alita; van Wering, Elisabeth R.; Westra, August H.; van de Loosdrecht, Arjan A.; de Jong, A.

    2012-01-01

    Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, an

  7. Identification of inflammatory cells in bovine milk by flow cytometry.

    Science.gov (United States)

    Redelman, D; Butler, S; Robison, J; Garner, D

    1988-09-01

    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.

  8. An overview of platelet indices and methods for evaluating platelet function in thrombocytopenic patients

    DEFF Research Database (Denmark)

    Vinholt, Pernille Just; Hvas, Anne-Mette; Nybo, Mads

    2014-01-01

    in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin and surface coverage by the Cone and Plate[let] analyser™ predict bleeding in selected thrombocytopenic populations...

  9. Modeling flow cytometry data for cancer vaccine immune monitoring.

    Science.gov (United States)

    Frelinger, Jacob; Ottinger, Janet; Gouttefangeas, Cécile; Chan, Cliburn

    2010-09-01

    Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories.

  10. In Vivo Flow Cytometry of Circulating Tumor-Associated Exosomes

    Directory of Open Access Journals (Sweden)

    Jacqueline Nolan

    2016-01-01

    Full Text Available Circulating tumor cells (CTCs demonstrated the potential as prognostic markers of metastatic development. However, the incurable metastasis can already be developed at the time of initial diagnosis with the existing CTC assays. Alternatively, tumor-associated particles (CTPs including exosomes can be a more valuable prognostic marker because they can be released from the primary tumor long before CTCs and in larger amount. However, little progress has been made in high sensitivity detection of CTPs, especially in vivo. We show here that in vivo integrated photoacoustic (PA and fluorescence flow cytometry (PAFFC platform can provide the detection of melanoma and breast-cancer-associated single CTPs with endogenously expressed melanin and genetically engineered proteins or exogenous dyes as PA and fluorescent contrast agents. The two-beam, time-of-light PAFFC can measure the sizes of CTCs and CTPs and identify bulk and rolling CTCs and CTC clusters, with no influence on blood flow instability. This technique revealed a higher concentration of CTPs than CTCs at an early cancer stage. Because a single tumor cell can release many CTPs and in vivo PAFFC can examine the whole blood volume, PAFFC diagnostic platform has the potential to dramatically improve (up to 105-fold the sensitivity of cancer diagnosis.

  11. Reevaluating multicolor flow cytometry to assess microbial viability.

    Science.gov (United States)

    Buysschaert, Benjamin; Byloos, Bo; Leys, Natalie; Van Houdt, Rob; Boon, Nico

    2016-11-01

    Flow cytometry is a rapid and quantitative method to determine bacterial viability. Although different stains can be used to establish viability, staining protocols are inconsistent and lack a general optimization approach. Very few "true" multicolor protocols, where dyes are combined in one sample, have been developed for microbiological applications. In this mini-review, the discrepancy between protocols for cell-permeant nucleic acid and functional stains are discussed as well as their use as viability dyes. Furthermore, optimization of staining protocols for a specific setup are described. Original data using the red-excitable SYTO dyes SYTO 59 to 64 and SYTO 17, combined with functional stains, for double and triple staining applications is also included. As each dye and dye combination behaves differently within a certain combination of medium matrix, microorganism, and instrument, protocols need to be tuned to obtain reproducible results. Therefore, single, double, and triple stains are reviewed, including the different parameters that influence staining such as stain kinetics, optimal stain concentration, and the effect of the chelator EDTA as membrane permeabilizer. In the last section, we highlight the need to investigate the stability of multicolor assays to ensure correct results as multiwell autoloaders are now commonly used.

  12. Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes

    Science.gov (United States)

    Della Porta, Matteo G.; Picone, Cristina

    2017-01-01

    The pathological hallmark of myelodysplastic syndromes (MDS) is marrow dysplasia, which represents the basis of the WHO classification of these disorders. This classification provides clinicians with a useful tool for defining the different subtypes of MDS and individual prognosis. The WHO proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). Therefore, there is clearly need to refine the accuracy to detect marrow dysplasia. Flow cytometry (FCM) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. The rationale for the application of FCM in the diagnostic work up of MDS is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that MDS have been found to have abnormal expression of several cellular antigens. To become applicable in clinical practice, FCM analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians. In this review, we discuss the most relevant progresses in detection of marrow dysplasia by FCM in MDS

  13. Detection of circulating breast cancer cells using photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran

    According to the American Cancer Society, more than 200,000 new cases of breast cancer are expected to be diagnosed this year. Moreover, about 40,000 women died from breast cancer last year alone. As breast cancer progresses in an individual, it can transform from a localized state to a metastatic one with multiple tumors distributed through the body, not necessarily contained within the breast. Metastasis is the spread of cancer through the body by circulating tumor cells (CTCs) which can be found in the blood and lymph of the diagnosed patient. Diagnosis of a metastatic state by the discovery of a secondary tumor can often come too late and hence, significantly reduce the patient's chance of survival. There is a current need for a CTC detection method which would diagnose metastasis before the secondary tumor occurs or reaches a size resolvable by current imaging systems. Since earlier detection would improve prognosis, this study proposes a method of labeling of breast cancer cells for detection with a photoacoustic flow cytometry system as a model for CTC detection in human blood. Gold nanoparticles and fluorescent polystyrene nanoparticles are proposed as contrast agents for T47D, the breast cancer cell line of choice. The labeling, photoacoustic detection limit, and sensitivity are first characterized and then applied to a study to show detection from human blood.

  14. Impact of cryopreservation on tetramer, cytokine flow cytometry, and ELISPOT

    Directory of Open Access Journals (Sweden)

    Morse Michael A

    2005-07-01

    Full Text Available Abstract Background Cryopreservation of PBMC and/or overnight shipping of samples are required for many clinical trials, despite their potentially adverse effects upon immune monitoring assays such as MHC-peptide tetramer staining, cytokine flow cytometry (CFC, and ELISPOT. In this study, we compared the performance of these assays on leukapheresed PBMC shipped overnight in medium versus cryopreserved PBMC from matched donors. Results Using CMV pp65 peptide pool stimulation or pp65 HLA-A2 tetramer staining, there was significant correlation between shipped and cryopreserved samples for each assay (p ≤ 0.001. The differences in response magnitude between cryopreserved and shipped PBMC specimens were not significant for most antigens and assays. There was significant correlation between CFC and ELISPOT assay using pp65 peptide pool stimulation, in both shipped and cryopreserved samples (p ≤ 0.001. Strong correlation was observed between CFC (using HLA-A2-restricted pp65 peptide stimulation and tetramer staining (p Conclusion We conclude that all three assays show concordant results on shipped versus cryopreserved specimens, when using a peptide-based readout. The assays are also concordant with each other in pair wise comparisons using equivalent antigen systems.

  15. Impedance Flow Cytometry: A Novel Technique in Pollen Analysis.

    Science.gov (United States)

    Heidmann, Iris; Schade-Kampmann, Grit; Lambalk, Joep; Ottiger, Marcel; Di Berardino, Marco

    2016-01-01

    An efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen. Developing and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays. Different stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population. The presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

  16. Detecting endotoxin with a flow cytometry-based magnetic aptasensor.

    Science.gov (United States)

    Zuo, Ming-Yan; Chen, Li-Juan; Jiang, Hao; Tan, Lin; Luo, Zhao-Feng; Wang, Yan-Mei

    2014-12-01

    Endotoxin, which is also known as lipopolysaccharide (LPS), is a marker for intruding gram-negative pathogens. It is essential to detect endotoxin quickly and sensitively in a complex milieu. A new flow cytometry (FCM)-based magnetic aptasensor assay that employs two endotoxin-binding aptamers and magnetic beads has been developed to detect endotoxin. The endotoxin-conjugated sandwich complex on magnetic beads was observed by scanning confocal laser microscopy. The resulting magnetic aptasensor rapidly detected (endotoxin within a broad dynamic detection range of 10(-8) to 10(0)mg/ml in the presence of bovine serum albumin (BSA), RNA, sucrose, and glucose, which are most likely to coexist with endotoxin in the majority of biological liquids. Only 2 μl of magnetic aptasensor was required to quantify the endotoxin solution. Furthermore, the magnetic aptasensor could be regenerated seven times and still presented an outstanding response to the endotoxin solution. Therefore, the magnetic aptasensor exhibited high sensitivity, selectivity, and reproducibility, thereby serving as a powerful tool for the quality control and high-throughput detection of endotoxin in the food and pharmaceutical industries.

  17. Applications of flow cytometry in environmental microbiology and biotechnology.

    Science.gov (United States)

    Bergquist, Peter L; Hardiman, Elizabeth M; Ferrari, Belinda C; Winsley, Tristrom

    2009-05-01

    Flow cytometry (FCM) is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It uses the principles of light scattering, light excitation and the emission from fluorescent molecules to generate specific multiparameter data from particles and cells. The cells are hydrodynamically focussed in a sheath solution before being intercepted by a focused light source provided by a laser. FCM has been used primarily in medical applications but is being used increasingly for the examination of individual cells from environmental samples. It has found uses in the isolation of both culturable and hitherto non-culturable bacteria present infrequently in environmental samples using appropriate growth conditions. FCM lends itself to high-throughput applications in directed evolution for the analysis of single cells or cell populations carrying mutant genes. It is also suitable for encapsulation studies where individual bacteria are compartmentalised with substrate in water-in-oil-in-water emulsions or with individual genes in transcriptional/translational mixtures for the production of mutant enzymes. The sensitivity of the technique has allowed the examination of gene optimisation by a procedure known as random or neutral drift where screening and selection is based on the retention of some predetermined level of activity through multiple rounds of mutagenesis.

  18. Viability staining of Cryptosporidium oocysts and Giardia cysts combined with flow cytometry

    NARCIS (Netherlands)

    Schets FM; Medema GJ; MGB

    1998-01-01

    The incorporation of flow cytometry as an additional purification step has improved the detection method for Cryptosporidium oocysts and Giardia cysts in water. Flow cytometry allows separation of (oo)cysts from interfering debris particles present in water concentrates and thus enables the applicat

  19. Role of flow cytometry in the diagnosis and monitoring of primary immunodeficiency disease.

    Science.gov (United States)

    O'gorman, Maurice R G

    2007-09-01

    This presentation is organized according to the recent classification of primary immunodeficiencies published by the International Union of Immunological Societies Primary Immunodeficiency meeting. The diseases have been classified into eight groups. After each list, individual diseases that are amenable to assessment by flow cytometry are reviewed with a brief clinical description and a discussion of the appropriate flow cytometry application.

  20. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

    NARCIS (Netherlands)

    Rawstron, Andy C.; Orfao, Alberto; Beksac, Meral; Bezdickova, Ludmila; Broolmans, Rik A.; Bumbea, Horia; Dalva, Klara; Fuhler, Gwenny; Gratama, Jan; Hose, Dirk; Kovarova, Lucie; Lioznov, Michael; Mateo, Gema; Morilla, Ricardo; Mylin, Anne K.; Omede, Paola; Pellat-Deceunynck, Catherine; Andres, Martin Perez; Petrucci, Maria; Ruggeri, Marina; Rymkiewicz, Grzegorz; Schmitz, Alexander; Schreder, Martin; Seynaeve, Carine; Spacek, Martin; de Tute, Ruth M.; Van Valckenborgh, Els; Weston-Bell, Nicky; Owen, Roger G.; Miguel, Jesus F. San; Sonneveld, Pieter; Johnsen, Hans E.

    2008-01-01

    The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating labora

  1. Viability staining of Cryptosporidium oocysts and Giardia cysts combined with flow cytometry

    NARCIS (Netherlands)

    Schets FM; Medema GJ; MGB

    1998-01-01

    Flow cytometrie als extra zuiveringsstap heeft de detectiemethode voor Cryptosporidium oocysten en Giardia cysten in water verbeterd. Door middel van flow cytometrie kunnen (oo)cysten gescheiden worden van het meeste debris dat in waterconcentraten aanwezig is, waardoor de toepassing van fluorogene

  2. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

    NARCIS (Netherlands)

    Rawstron, Andy C.; Orfao, Alberto; Beksac, Meral; Bezdickova, Ludmila; Broolmans, Rik A.; Bumbea, Horia; Dalva, Klara; Fuhler, Gwenny; Gratama, Jan; Hose, Dirk; Kovarova, Lucie; Lioznov, Michael; Mateo, Gema; Morilla, Ricardo; Mylin, Anne K.; Omede, Paola; Pellat-Deceunynck, Catherine; Andres, Martin Perez; Petrucci, Maria; Ruggeri, Marina; Rymkiewicz, Grzegorz; Schmitz, Alexander; Schreder, Martin; Seynaeve, Carine; Spacek, Martin; de Tute, Ruth M.; Van Valckenborgh, Els; Weston-Bell, Nicky; Owen, Roger G.; Miguel, Jesus F. San; Sonneveld, Pieter; Johnsen, Hans E.

    2008-01-01

    The European Myeloma Network (EMN) organized two flow cytometry workshops. The first aimed to identify specific indications for flow cytometry in patients with monoclonal gammopathies, and consensus technical approaches through a questionnaire-based review of current practice in participating labora

  3. Ultrafast quantitative time-stretch imaging flow cytometry of phytoplankton

    Science.gov (United States)

    Lai, Queenie T. K.; Lau, Andy K. S.; Tang, Anson H. L.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2016-03-01

    Comprehensive quantification of phytoplankton abundance, sizes and other parameters, e.g. biomasses, has been an important, yet daunting task in aquatic sciences and biofuel research. It is primarily because of the lack of effective tool to image and thus accurately profile individual microalgae in a large population. The phytoplankton species are highly diversified and heterogeneous in terms of their sizes and the richness in morphological complexity. This fact makes time-stretch imaging, a new ultrafast real-time optical imaging technology, particularly suitable for ultralarge-scale taxonomic classification of phytoplankton together with quantitative image recognition and analysis. We here demonstrate quantitative imaging flow cytometry of single phytoplankton based on quantitative asymmetric-detection time-stretch optical microscopy (Q-ATOM) - a new time-stretch imaging modality for label-free quantitative phase imaging without interferometric implementations. Sharing the similar concept of Schlieren imaging, Q-ATOM accesses multiple phase-gradient contrasts of each single phytoplankton, from which the quantitative phase profile is computed. We employ such system to capture, at an imaging line-scan rate of 11.6 MHz, high-resolution images of two phytoplankton populations (scenedesmus and chlamydomonas) in ultrafast microfluidic flow (3 m/s). We further perform quantitative taxonomic screening analysis enabled by this technique. More importantly, the system can also generate quantitative phase images of single phytoplankton. This is especially useful for label-free quantification of biomasses (e.g. lipid droplets) of the particular species of interest - an important task adopted in biofuel applications. Combining machine learning for automated classification, Q-ATOM could be an attractive platform for continuous and real-time ultralarge-scale single-phytoplankton analysis.

  4. Detection of CFTR protein in human leukocytes by flow cytometry.

    Science.gov (United States)

    Johansson, Jan; Vezzalini, Marzia; Verzè, Genny; Caldrer, Sara; Bolognin, Silvia; Buffelli, Mario; Bellisola, Giuseppe; Tridello, Gloria; Assael, Baroukh Maurice; Melotti, Paola; Sorio, Claudio

    2014-07-01

    Leukocytes have previously been shown to express detectable levels of the protein cystic fibrosis transmembrane conductance regulator (CFTR). This study aims to evaluate the application of flow cytometric (FC) analysis to detect CFTR expression, and changes thereof, in these cells. Aliquots (200 μL) of peripheral whole blood from 12 healthy control volunteers (CTRLs), 12 carriers of a CFTR mutation (CFC), and 40 patients with cystic fibrosis (CF) carrying various combinations of CFTR mutations were incubated with specific fluorescent probes recognizing CFTR protein expressed on the plasma membrane of leukocytes. FC was applied to analyze CFTR expression in monocytes, lymphocytes, and polymorphonuclear (PMN) cells. CFTR protein was detected in monocytes and lymphocytes, whereas inconclusive results were obtained from the analysis of PMN cells. Mean fluorescence intensity (MFI) ratio value and %CFTR-positive cells above a selected threshold were the two parameters selected to quantify CFTR expression in cells. Lowest variability and the highest reproducibility were obtained when analyzing monocytes. ANOVA results indicated that both parameters were able to discriminate monocytes of healthy controls and CF individuals according to CFTR mutation classes with high accuracy. Significantly increased MFI ratio values were recorded in CFTR-defective cells that were also able to improve CFTR function after ex vivo treatment with PTC124 (Ataluren), an investigative drug designed to permit the ribosome to read through nonsense CFTR mutations. The method described is minimally invasive and may be used in the monitoring of responses to drugs whose efficacy can depend on increased CFTR protein expression levels. © 2014 International Society for Advancement of Cytometry.

  5. Performance of calibration standards for antigen quantitation with flow cytometry.

    Science.gov (United States)

    Lenkei, R; Gratama, J W; Rothe, G; Schmitz, G; D'hautcourt, J L; Arekrans, A; Mandy, F; Marti, G

    1998-10-01

    In the frame of the activities initiated by the Task Force for Antigen Quantitation of the European Working Group on Clinical Cell Analysis (EWGCCA), an experiment was conducted to evaluate microbead standards used for quantitative flow cytometry (QFCM). An unified window of analysis (UWA) was established on three different instruments (EPICS XL [Coulter Corporation, Miami, FL], FACScan and FACS Calibur [Becton Dickinson, San Jose, CA]) with QC3 microbeads (FCSC, PR). By using this defined fluorescence intensity scale, the performance of several monoclonal antibodies directed to CD3, CD4, and CD8 (conjugated and unconjugated), from three manufacturers (BDIS, Coulter [Immunotech], and DAKO) was tested. In addition, the QIFI system (DAKO) and QuantiBRITE (BDIS), and a method of relative fluorescence intensity (RFI, method of Giorgi), were compared. mAbs reacting with three more antigens, CD16, CD19, and CD38 were tested on the FACScan instrument. Quantitation was carried out using a single batch of cryopreserved peripheral blood leukocytes, and all tests were performed as single color analyses. Significant correlations were observed between the antibody-binding capacity (ABC) values of the same CD antigen measured with various calibrators and with antibodies differing in respect to vendor, labeling and possible epitope recognition. Despite the significant correlations, the ABC values of most monoclonal antibodies differed by 20-40% when determined by the different fluorochrome conjugates and different calibrators. The results of this study indicate that, at the present stage of QFCM consistent ABC values may be attained between laboratories provided that a specific calibration system is used including specific calibrators, reagents, and protocols.

  6. Understanding microbial/DOM interactions using fluorescence and flow cytometry

    Science.gov (United States)

    Fox, Bethany; Rushworth, Cathy; Attridge, John; Anesio, Alexandre; Cox, Tim; Reynolds, Darren

    2015-04-01

    The transformation and movement of dissolved organic carbon (DOC) within freshwater aquatic systems is an important factor in the global cycling of carbon. DOC within aquatic systems is known to underpin the microbial food web and therefore plays an essential role in supporting and maintaining the aquatic ecosystem. Despite this the interactions between bacteria and dissolved organic matter (DOM) are not well understood, although the literature indicates that the microbial processing of bioavailable DOM is essential during the production of autochthonous, labile, DOM. DOM can be broadly characterised by its fluorescing properties and Coble et al. (2014) define terrestrially derived DOM as exhibiting "peak C" fluorescence, whilst labile microbially derived DOM is defined as showing "peak T" fluorescence. Our work explores the microbial/DOM interactions by analysing aquatic samples using fluorescence excitation and emission matrices (EEMs) in conjunction with microbial consumption of dissolved oxygen. Environmental and synthetic water samples were subjected to fluorescence characterisation using both fluorescence spectroscopy and in situ fluorescence sensors (Chelsea Technologies Group Ltd.). PARAFAC analysis and peak picking were performed on EEMs and compared with flow cytometry data, used to quantify bacterial numbers present within samples. Synthetic samples were created using glucose, glutamic acid, nutrient-rich water and a standard bacterial seed. Synthetic samples were provided with terrestrially derived DOM via the addition of an aliquot of environmental water. Using a closed system approach, samples were incubated over time (up to a maximum of 20 days) and analysed at pre-defined intervals. The main focus of our work is to improve our understanding of microbial/DOM interactions and how these interactions affect both the DOM characteristics and microbial food web in freshwater aquatic systems. The information gained, in relation to the origin, microbial

  7. Flow Cytometry of the Side Population: Tips & Tricks

    Directory of Open Access Journals (Sweden)

    Irene Sales-Pardo

    2006-01-01

    Full Text Available Background: The Side Population (SP has become an important hallmark for the definition of the stem cell compartment, especially in the detection of these cells and in their physical isolation by fluorescence-activated cell sorting (FACS. SP cells are CD34neg and were discovered using ultraviolet excitation based on the efflux of Hoechst 33342 (Ho342. Although the method works as originally described, we believe that this method is difficult for most investigators. First, because the ability to discriminate SP cells is based on the differential retention of Ho342 during a functional assay; second, because of the difficulties in setting the right experimental and acquisition conditions; and third, because the analysis of the acquired data requires an extensive expertise on flow cytometry to accurately detect the SP events. Methods: First of all and mainly for the SP application, the laser beam paths were exhaustively checked to ensure the lowest coefficients of variation. Blood suspensions were prepared by erythrocyte lysis with ammonium chloride and hematopoietic cells were labeled with Ho342. Results: The Ho342 concentration and the staining procedure are critical for the optimal resolution of the SP cells. Although UV laser alignment is very important to resolve the dim tail that outlines the SP, the problem with Ho342 excitation is not the Hoechst Blue emission, but rather the Hoechst Red's (because of the weak emission. Conclusions: Each laboratory must establish its own expected ranges based on its instrument and results may vary slightly due to instrument differences such as the narrowness of the band pass filters, laser power, laser emission wavelength, nozzle type, differential of pressure, light collection system (cuvette versus jet-in-air and beam shaping optics.

  8. Impact of the new Beckman Coulter Cytomics FC 500 5-color flow cytometer on a regional flow cytometry clinical laboratory service.

    Science.gov (United States)

    Luider, J; Cyfra, M; Johnson, P; Auer, I

    2004-01-01

    Calgary Laboratory Services (CLS) in Alberta, Canada, is the regional reference laboratory providing flow cytometry services for southern Alberta and southeastern British Columbia. As a busy reference flow laboratory we provide flow cytometry immunophenotyping for investigation and diagnosis of acute and chronic leukemias, lymphomas, immunodeficiencies, neuroblastoma, platelet disorders, and interstitial lung disease (ILD). Because of increasing workload and the continual effort to improve the service to our health care providers, CLS invested in the new Beckman Coulter Cytomics FC 500 5-color flow cytometer. In addition to time and labor savings due to reduced maintenance and operating system design, this new flow cytometer automates many of the previous manual steps involved in quality control and flow cytometric analysis. It also incorporates 2 lasers and is capable of measuring 5-color antibody combinations in a single tube, enabling us to reduce the number of tubes and overall costs, giving us better gating options for minimal residual disease analysis. We present the first published evaluation, an assessment of the overall productivity and cost impact of the new state-of-the-art Cytomics FC 500 flow cytometer. Implementation of the Cytomics FC 500 has resulted in a 20% reduction in reagent costs and shorter turnaround time for analysis and diagnosis. This instrument has allowed us to reduce our acute leukemia panel from 17 to 13 tubes, our lymphoma panel from 13 to 7 tubes, and our ILD panel from 4 to 2 tubes. The availability of 2 lasers provides more flexibility in choosing antibodies and conjugates to customize immunophenotyping panels. It also allows us to use the DRAQ5 dye and simultaneously analyze the immunophenotype and DNA content of cells with very little compensation. Many of the arduous, time-consuming flow operator tasks often associated with previous generation flow cytometry instruments, such as color compensation, list mode analysis, sample

  9. [Which technique should be used in the phenotyping of lymphocytic alveolitis: Immunocytochemistry or flow cytometry].

    Science.gov (United States)

    Mlika, Mona; Kasmi, Rihem; Safra, Ines; Braham, Emna; Chebbi, Chokri; Mezni, Faouzi El

    2017-09-18

    Diffuse interstitial pneumonias are considered as a group of multiple affections characterized by challenging diagnoses because of the lack of specific clinical signs. Radiologic investigations highlight the diagnoses in most of the cases but bronchoalveolar lavage plays a key role in the diagnostic diagram. We aim to compare the immunocytochemical technique and the flow cytometry in the phenotyping of lymphocytic alveolitis. We described a series of 32 lymphocytic alveolitis, which were analyzed using immunocytochemistry and flow cytometry. We found a good reproducibility between the immunocytochemistry performed on smears and cytoblocks (kappa=0.7) and a poor reproducibility between immunocytochemistry and flow cytometry (kappa=0.35). Our study emphasized on the poor reproducibility between immunocytochemistry and flow cytometry. Further studies about the reliability of both techniques are needed especially in discordant cases. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  10. A comparison of flow cytometry and immunohistochemistry in human colorectal cancers

    DEFF Research Database (Denmark)

    Diederichsen, Axel Cosmus Pyndt; Hansen, T P; Nielsen, O

    1998-01-01

    In human colorectal cancer it has been reported that some tumours lack the HLA-ABC antigens. This has been interpreted as reflecting tumour escape from the immune system. Earlier data have been obtained by immunohistochemistry. In this study, we compared the expression of HLA-ABC, HLA-DR, CD80 (B7-1......) and CD54 (ICAM-1) in 20 tumours using both a conventional immunohistochemistry two-layer technique and multiparameter flow cytometry, gating on an epithelial cell marker. Colorectal cancer tissue used in flow cytometry was dissociated with collagenase, deoxyribonuclease and hyaluronidase. The intensity...... was superior to immunohistochemistry in 33 out of 80 cases, and showed that tumours described as HLA-ABC negative by immunohistochemistry were in fact weakly positive for HLA-ABC. We conclude that flow cytometry and immunohistochemistry are complementary, and that flow cytometry is superior...

  11. Implementation of erythroid lineage analysis by flow cytometry in diagnostic models for myelodysplastic syndromes

    Science.gov (United States)

    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.

    2017-01-01

    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 www.trialregister.nl as NTR1825; EudraCT n.: 2008-002195-10 PMID:27658438

  12. Determination of total bacterial count in raw milk by flow cytometry

    OpenAIRE

    Dubravka Samaržija; Neven Antunac; Tomislav Pogačić; Sanja Sikora

    2004-01-01

    The automatic flow cytometry as routine method for total bacterial count determination of raw ex-farm milk has recently been accepted in Croatia. This method significantly differs from the reference method (Standard Plate Count) mostly in the presentation of the results obtained. Therefore, this paper summarized experiences in the application of flow cytometry in the dairy laboratories practice. The principle and the practice of the method, methodological details and factors influencing the r...

  13. Comparative exploration of multidimensional flow cytometry software: a model approach evaluating T cell polyfunctional behavior.

    Science.gov (United States)

    Spear, Timothy T; Nishimura, Michael I; Simms, Patricia E

    2017-08-01

    Advancement in flow cytometry reagents and instrumentation has allowed for simultaneous analysis of large numbers of lineage/functional immune cell markers. Highly complex datasets generated by polychromatic flow cytometry require proper analytical software to answer investigators' questions. A problem among many investigators and flow cytometry Shared Resource Laboratories (SRLs), including our own, is a lack of access to a flow cytometry-knowledgeable bioinformatics team, making it difficult to learn and choose appropriate analysis tool(s). Here, we comparatively assess various multidimensional flow cytometry software packages for their ability to answer a specific biologic question and provide graphical representation output suitable for publication, as well as their ease of use and cost. We assessed polyfunctional potential of TCR-transduced T cells, serving as a model evaluation, using multidimensional flow cytometry to analyze 6 intracellular cytokines and degranulation on a per-cell basis. Analysis of 7 parameters resulted in 128 possible combinations of positivity/negativity, far too complex for basic flow cytometry software to analyze fully. Various software packages were used, analysis methods used in each described, and representative output displayed. Of the tools investigated, automated classification of cellular expression by nonlinear stochastic embedding (ACCENSE) and coupled analysis in Pestle/simplified presentation of incredibly complex evaluations (SPICE) provided the most user-friendly manipulations and readable output, evaluating effects of altered antigen-specific stimulation on T cell polyfunctionality. This detailed approach may serve as a model for other investigators/SRLs in selecting the most appropriate software to analyze complex flow cytometry datasets. Further development and awareness of available tools will help guide proper data analysis to answer difficult biologic questions arising from incredibly complex datasets. © Society

  14. PLATELET ADHESION TO POLYURETHANE UREA UNDER PULSATILE FLOW CONDITIONS

    Science.gov (United States)

    Navitsky, Michael A.; Taylor, Joshua O.; Smith, Alexander B.; Slattery, Margaret J.; Deutsch, Steven; Siedlecki, Christopher A.; Manning, Keefe B.

    2014-01-01

    Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s−1. The aim of the current work is to determine platelet adhesion properties to the polyurethane urea surface as a function of time varying shear exposure. A rotating disk system is used to study the influence of steady and pulsatile flow conditions (e.g. cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments retain the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk is rotated in platelet rich bovine plasma for two hours with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow is found to exponentially decay with increasing shear rate. Adhesion levels are found to depend upon peak platelet flux and shear rate regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices. PMID:24721222

  15. Platelet adhesion to polyurethane urea under pulsatile flow conditions.

    Science.gov (United States)

    Navitsky, Michael A; Taylor, Joshua O; Smith, Alexander B; Slattery, Margaret J; Deutsch, Steven; Siedlecki, Christopher A; Manning, Keefe B

    2014-12-01

    Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s(-1). The aim of the current work is to determine the properties of platelet adhesion to the polyurethane urea surface as a function of time-varying shear exposure. A rotating disk system was used to study the influence of steady and pulsatile flow conditions (e.g., cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments were conducted with the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk was rotated in platelet-rich bovine plasma for 2 h, with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow was found to decay exponentially with increasing shear rate. Adhesion levels were found to depend upon peak platelet flux and shear rate, regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices.

  16. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Directory of Open Access Journals (Sweden)

    Vendula Pospichalova

    2015-03-01

    Full Text Available Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm, their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE and/or lipid- (FM specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the

  17. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer.

    Science.gov (United States)

    Pospichalova, Vendula; Svoboda, Jan; Dave, Zankruti; Kotrbova, Anna; Kaiser, Karol; Klemova, Dobromila; Ilkovics, Ladislav; Hampl, Ales; Crha, Igor; Jandakova, Eva; Minar, Lubos; Weinberger, Vit; Bryja, Vitezslav

    2015-01-01

    Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine

  18. Flow cytometry susceptibility testing for conventional antifungal drugs and Comparison with the NCCLS Broth Macrodilution Test

    Directory of Open Access Journals (Sweden)

    M.J. Najafzadeh

    2009-08-01

    Full Text Available Introduction: During the last decade, the incidence of fungal infection has been increased in many countries. Because of the advent of resistant to antifungal agents, determination of an efficient strategic plan for treatment of fungal disease is an important issue in clinical mycology. Many methods have been introduced and developed for determination of invitro susceptibility tests. During the recent years, flow cytometry has developed to solving the problem and many papers have documented the usefulness of this technique. Materials and methods: As the first step, the invitro susceptibility of standard PTCC (Persian Type of Culture Collection strain and some clinical isolates of Candida consisting of Candida albicans, C. dubliniensis, C. glabrata, C. kefyer and C. parapsilosis were evaluated by macrodilution broth method according to NCCLS (National Committee for Clinical Laboratory Standards guidelines and flow cytometry susceptibility test. Results:  The data indicated that macro dilution broth methods and flow cytometry have the same results in determination of MIC (Minimum Inhibitory Concentration for amphotericin B, clotrimazole, fluconazole, ketoconazole and miconazole in C. albicans PTCC 5027 as well as clinical Candida isolates, such as C.albicans, C.dubliniensis, C.glabrata C.kefyr, and C.parapsilosis. Discussion: Comparing the results obtained by macrodilution broth and flow cytometry methods revealed that flow cytometry was faster. It is suggested that flow cytometry susceptibility test can be used as a powerful tool for determination of MIC and administration of the best antifungal drug in treatment of patients with Candida infections.

  19. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    Science.gov (United States)

    Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

  20. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification

    Science.gov (United States)

    Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple perform...

  1. Multiplexed microbead immunoassays by flow cytometry for molecular profiling: Basic concepts and proteomics applications.

    Science.gov (United States)

    Krishhan, V V; Khan, Imran H; Luciw, Paul A

    2009-01-01

    Flow cytometry was originally established as an automated method for measuring optical or fluorescence characteristics of cells or particles in suspension. With the enormous increase in development of reliable electronics, lasers, micro-fluidics, as well as many advances in immunology and other fields, flow cytometers have become user-friendlier, less-expensive instruments with an increasing importance for both basic research and clinical applications. Conventional uses of flow cytometry include immunophenotyping of blood cells and the analysis of the cell cycle. Importantly, methods for labeling microbeads with unique combinations of fluorescent spectral signatures have made multiplex analysis of soluble analytes (i.e. the ability to detect multiple targets in a single test sample) feasible by flow cytometry. The result is a rapid, high-throughput, sensitive, and reproducible detection technology for a wide range of biomedical applications requiring detection of proteins (in cells and biofluids) and nucleic acids. Thus, novel methods of flow cytometry are becoming important for diagnostic purposes (e.g. identifying multiple clinical biomarkers for a wide range of diseases) as well as for developing novel therapies (e.g. elucidating drug mechanisms and potential toxicities). In addition, flow cytometry for multiplex analysis, coupled with automated sample handling devices, has the potential to significantly enhance proteomics research, particularly analysis of post-translational modifications of proteins, on a large scale. Inherently, flow cytometry methods are strongly rooted in the laws of the physics of optics, fluidics, and electromagnetism. This review article describes principles and early sources of flow cytometry, provides an introduction to the multiplex microbead technology, and discusses its applications and advantages in comparison to other methods. Anticipated future directions, particularly for translational research in medicine, are also discussed.

  2. FuGEFlow: data model and markup language for flow cytometry.

    Science.gov (United States)

    Qian, Yu; Tchuvatkina, Olga; Spidlen, Josef; Wilkinson, Peter; Gasparetto, Maura; Jones, Andrew R; Manion, Frank J; Scheuermann, Richard H; Sekaly, Rafick-Pierre; Brinkman, Ryan R

    2009-06-16

    Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt) standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE) formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. We used the MagicDraw modelling tool to design a UML model (Flow-OM) according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML). We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow), which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets. Additional project documentation, including

  3. FuGEFlow: data model and markup language for flow cytometry

    Directory of Open Access Journals (Sweden)

    Manion Frank J

    2009-06-01

    Full Text Available Abstract Background Flow cytometry technology is widely used in both health care and research. The rapid expansion of flow cytometry applications has outpaced the development of data storage and analysis tools. Collaborative efforts being taken to eliminate this gap include building common vocabularies and ontologies, designing generic data models, and defining data exchange formats. The Minimum Information about a Flow Cytometry Experiment (MIFlowCyt standard was recently adopted by the International Society for Advancement of Cytometry. This standard guides researchers on the information that should be included in peer reviewed publications, but it is insufficient for data exchange and integration between computational systems. The Functional Genomics Experiment (FuGE formalizes common aspects of comprehensive and high throughput experiments across different biological technologies. We have extended FuGE object model to accommodate flow cytometry data and metadata. Methods We used the MagicDraw modelling tool to design a UML model (Flow-OM according to the FuGE extension guidelines and the AndroMDA toolkit to transform the model to a markup language (Flow-ML. We mapped each MIFlowCyt term to either an existing FuGE class or to a new FuGEFlow class. The development environment was validated by comparing the official FuGE XSD to the schema we generated from the FuGE object model using our configuration. After the Flow-OM model was completed, the final version of the Flow-ML was generated and validated against an example MIFlowCyt compliant experiment description. Results The extension of FuGE for flow cytometry has resulted in a generic FuGE-compliant data model (FuGEFlow, which accommodates and links together all information required by MIFlowCyt. The FuGEFlow model can be used to build software and databases using FuGE software toolkits to facilitate automated exchange and manipulation of potentially large flow cytometry experimental data sets

  4. In vitro micronucleus assay for the analysis of total particulate matter in cigarette smoke: comparison of flow cytometry and laser scanning cytometry with microscopy.

    Science.gov (United States)

    Yao, Jianhua; Gao, Qian; Mi, Qili; Li, Xuemei; Miao, Mingming; Cheng, Peng; Luo, Ying

    2013-08-15

    The possible genotoxicity of the total particulate matter (TPM) in cigarette smoke has typically been evaluated using the in vitro micronucleus assay. In recent years, automated scoring techniques have been developed to replace the manual counting process in this assay. However, these automated scoring techniques have not been applied in routine genotoxicity assays for the analysis of TPM to improve the assay efficiency. Chinese hamster ovary (CHO) cells were treated with TPM produced from 14 types of cigarettes at five concentrations (25-200μg/ml) without exogenous metabolic activation. The three following methods were used to score the micronucleus (MN) frequency: (a) flow cytometry with SYTOX and EMA dyes, which differentially stain micronuclei and apoptotic/necrotic chromatin to enhance assay reliability; (b) laser scanning cytometry with FITC and PI dyes, which is a system that combines the analytical capabilities of flow and image cytometry; and (c) visual microcopy with Giemsa dye. The test results obtained using the three methods were compared using correlation analysis. The key findings for this set of compounds include the following: (a) both flow cytometry- and laser scanning cytometry-based methods were effective for MN identification, (b) the three scoring methods could detect dose-dependent micronucleus formation for the 14 types of TPM, and (c) the MN frequencies that were measured in the same samples by flow cytometry, laser scanning cytometry, and visual microscopy were highly correlated, and there were no significant differences (p>0.05). In conclusion, both flow cytometry and laser scanning cytometry can be used to evaluate the MN frequency induced by TPM without exogenous metabolic activation. The simpler and faster processing and the high correlation of the results make these two automatic methods appropriate tools for use in in vitro micronucleus assays for the analysis of TPM using CHO cells.

  5. flowClust: a Bioconductor package for automated gating of flow cytometry data

    Directory of Open Access Journals (Sweden)

    Lo Kenneth

    2009-05-01

    Full Text Available Abstract Background As a high-throughput technology that offers rapid quantification of multidimensional characteristics for millions of cells, flow cytometry (FCM is widely used in health research, medical diagnosis and treatment, and vaccine development. Nevertheless, there is an increasing concern about the lack of appropriate software tools to provide an automated analysis platform to parallelize the high-throughput data-generation platform. Currently, to a large extent, FCM data analysis relies on the manual selection of sequential regions in 2-D graphical projections to extract the cell populations of interest. This is a time-consuming task that ignores the high-dimensionality of FCM data. Results In view of the aforementioned issues, we have developed an R package called flowClust to automate FCM analysis. flowClust implements a robust model-based clustering approach based on multivariate t mixture models with the Box-Cox transformation. The package provides the functionality to identify cell populations whilst simultaneously handling the commonly encountered issues of outlier identification and data transformation. It offers various tools to summarize and visualize a wealth of features of the clustering results. In addition, to ensure its convenience of use, flowClust has been adapted for the current FCM data format, and integrated with existing Bioconductor packages dedicated to FCM analysis. Conclusion flowClust addresses the issue of a dearth of software that helps automate FCM analysis with a sound theoretical foundation. It tends to give reproducible results, and helps reduce the significant subjectivity and human time cost encountered in FCM analysis. The package contributes to the cytometry community by offering an efficient, automated analysis platform which facilitates the active, ongoing technological advancement.

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

    Science.gov (United States)

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

    1999-04-01

    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.

  7. Amphiphilic mediated sample preparation for micro-flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Clague, David S. (Livermore, CA); Wheeler, Elizabeth K. (Livermore, CA); Lee, Abraham P. (Irvine, CA)

    2009-03-17

    A flow cytometer includes a flow cell for detecting the sample, an oil phase in the flow cell, a water phase in the flow cell, an oil-water interface between the oil phase and the water phase, a detector for detecting the sample at the oil-water interface, and a hydrophobic unit operatively connected to the sample. The hydrophobic unit is attached to the sample. The sample and the hydrophobic unit are placed in an oil and water combination. The sample is detected at the interface between the oil phase and the water phase.

  8. A liposome-based size calibration method for measuring microvesicles by flow cytometry.

    Science.gov (United States)

    Simonsen, J B

    2016-01-01

    ESSENTIALS: A gold standard to determine the sizes of microvesicles by flow cytometry is needed. We used fluorescently labeled liposomes to estimate the size of microvesicles with flow cytometry. We suggest that liposomes are more accurate size calibrators than the commonly used polystyrene beads. The liposome-based size calibrators improve the size assessment of microvesicle made with flow cytometry. During the past years, the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. This work suggests that artificial vesicles can be used as calibrators to estimate the size of microvesicles from the side scattering (SSC) measured with flow cytometry. We prepared fluorescently labeled liposomes with different maximum sizes defined by the pore size (200, 400, 800, and 1000 nm) of the membrane used for the extrusion. The fluorescence strengths from the largest liposomes pertaining to each pore size enabled us to verify the correlation between the SSC from a liposome and the corresponding size. This study indicates that artificial vesicles are more accurate size calibrators compared to the commonly used polystyrene calibrator beads illustrated by the SSC from 110 nm polystyrene beads corresponds to the scattering from ~400 nm vesicle-like particles. We also show that this method of size assessment based on SSC has a low resolution that is roughly estimated to be between 60 and 200 nm, dependent on the vesicle size. © 2015 International Society on Thrombosis and Haemostasis.

  9. Use of flow cytometry for rapid and accurate enumeration of live pathogenic Leptospira strains.

    Science.gov (United States)

    Fontana, Célia; Crussard, Steve; Simon-Dufay, Nathalie; Pialot, Daniel; Bomchil, Natalia; Reyes, Jean

    2017-01-01

    Enumeration of Leptospira, the causative agent of leptospirosis, is arduous mainly because of its slow growth rate. Rapid and reliable tools for numbering leptospires are still lacking. The current standard for Leptospira cultures is the count on Petroff-Hausser chamber under dark-field microscopy, but this method remains time-consuming, requires well-trained operators and lacks reproducibility. Here we present the development of a flow-cytometry technique for counting leptospires. We showed that upon addition of fluorescent dyes, necessary to discriminate the bacterial population from debris, several live Leptospira strains could be enumerated at different physiologic states. Flow cytometry titers were highly correlated to counts with Petroff-Hausser chambers (R(2)>0.99). Advantages of flow cytometry lie in its rapidity, its reproducibility significantly higher than Petroff-Hausser method and its wide linearity range, from 10(4) to 10(8)leptospires/ml. Therefore, flow cytometry is a fast, reproducible and sensitive tool representing a promising technology to replace current enumeration techniques of Leptospira in culture. We were also able to enumerate Leptospira in artificially infected urine and blood with a sensitivity limit of 10(5)leptospires/ml and 10(6)leptospires/ml, respectively, demonstrating the feasibility to use flow cytometry as first-line tool for diagnosis or bacterial dissemination studies.

  10. Characterization of extracellular vesicles in whole blood: Influence of pre-analytical parameters and visualization of vesicle-cell interactions using imaging flow cytometry.

    Science.gov (United States)

    Fendl, Birgit; Weiss, René; Fischer, Michael B; Spittler, Andreas; Weber, Viktoria

    2016-09-09

    Extracellular vesicles are central players in intercellular communication and are released from the plasma membrane under tightly regulated conditions, depending on the physiological and pathophysiological state of the producing cell. Their heterogeneity requires a spectrum of methods for isolation and characterization, where pre-analytical parameters have profound impact on vesicle analysis, particularly in blood, since sampling, addition of anticoagulants, as well as post-sampling vesicle generation may influence the outcome. Here, we characterized microvesicles directly in whole blood using a combination of flow cytometry and imaging flow cytometry. We assessed the influence of sample agitation, anticoagulation, and temperature on post-sampling vesicle generation, and show that vesicle counts remained stable over time in samples stored without agitation. Storage with gentle rolling mimicking agitation, in contrast, resulted in strong release of platelet-derived vesicles in blood anticoagulated with citrate or heparin, whereas vesicle counts remained stable upon anticoagulation with EDTA. Using imaging flow cytometry, we could visualize microvesicles adhering to blood cells and revealed an anticoagulant-dependent increase in vesicle-cell aggregates over time. We demonstrate that vesicles adhere preferentially to monocytes and granulocytes in whole blood, while no microvesicles could be visualized on lymphocytes. Our data underscore the relevance of pre-analytical parameters in vesicle analysis and demonstrate that imaging flow cytometry is a suitable tool to study the interaction of extracellular vesicles with their target cells.

  11. Light-scattering polarization measurements as a new parameter in flow cytometry

    NARCIS (Netherlands)

    Grooth, de B.G.; Terstappen, L.W.M.M.; Puppels, G.J.; Greve, J.

    1987-01-01

    Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized

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

    Science.gov (United States)

    Fromm, Jonathan R; Wood, Brent L

    2012-07-01

    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.

  13. High-throughput autofluorescence flow cytometry of breast cancer metabolism (Conference Presentation)

    Science.gov (United States)

    Shah, Amy T.; Cannon, Taylor M.; Higginbotham, Jim N.; Skala, Melissa C.

    2016-02-01

    Tumor heterogeneity poses challenges for devising optimal treatment regimens for cancer patients. In particular, subpopulations of cells can escape treatment and cause relapse. There is a need for methods to characterize tumor heterogeneity of treatment response. Cell metabolism is altered in cancer (Warburg effect), and cells use the autofluorescent cofactor NADH in numerous metabolic reactions. Previous studies have shown that microscopy measurements of NADH autofluorescence are sensitive to treatment response in breast cancer, and these techniques typically assess hundreds of cells per group. An alternative approach is flow cytometry, which measures fluorescence on a single-cell level and is attractive for characterizing tumor heterogeneity because it achieves high-throughput analysis and cell sorting in millions of cells per group. Current applications for flow cytometry rely on staining with fluorophores. This study characterizes flow cytometry measurements of NADH autofluorescence in breast cancer cells. Preliminary results indicate flow cytometry of NADH is sensitive to cyanide perturbation, which inhibits oxidative phosphorylation, in nonmalignant MCF10A cells. Additionally, flow cytometry is sensitive to higher NADH intensity for HER2-positive SKBr3 cells compared with triple-negative MDA-MB-231 cells. These results agree with previous microscopy studies. Finally, a mixture of SKBr3 and MDA-MB-231 cells were sorted into each cell type using NADH intensity. Sorted cells were cultured, and microscopy validation showed the expected morphology for each cell type. Ultimately, flow cytometry could be applied to characterize tumor heterogeneity based on treatment response and sort cell subpopulations based on metabolic profile. These achievements could enable individualized treatment strategies and improved patient outcomes.

  14. Automation in high-content flow cytometry screening.

    Science.gov (United States)

    Naumann, U; Wand, M P

    2009-09-01

    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.

  15. Potential Use of Quantum Dots in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Raquel Ibáñez-Peral

    2008-12-01

    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.

  16. High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays

    DEFF Research Database (Denmark)

    Tiendrebeogo, Regis W; Adu, Bright; Singh, Susheel K

    2014-01-01

    BACKGROUND: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent...... distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge. METHODS: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45...

  17. Flow cytometry approach for studying the interaction between ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-06-29

    Jun 29, 2016 ... Thirty five bacteria isolates were obtained from arid soil in the south of Algeria. Three of ..... desolvation temperature, 250°C; nitrogen flow, 500 l/h; cone ..... Andean soils of Peru and their potential PGPR characteristic. Braz. J.

  18. A Novel Statistical Analysis and Interpretation of Flow Cytometry Data

    Science.gov (United States)

    2013-07-05

    death processes at the population level to the observed flu - orescence intensity profiles as measured by a flow cytometer (Figures 1 and 2). Because... Spanish Ministry of Science and Innovation. The authors are grateful to several referees for a number of helpful comments. References [1] J.E. Aubin

  19. iFlow: A Graphical User Interface for Flow Cytometry Tools in Bioconductor

    Directory of Open Access Journals (Sweden)

    Kyongryun Lee

    2009-01-01

    Full Text Available Flow cytometry (FCM has become an important analysis technology in health care and medical research, but the large volume of data produced by modern high-throughput experiments has presented significant new challenges for computational analysis tools. The development of an FCM software suite in Bioconductor represents one approach to overcome these challenges. In the spirit of the R programming language (Tree Star Inc., “FlowJo”, these tools are predominantly console-driven, allowing for programmatic access and rapid development of novel algorithms. Using this software requires a solid understanding of programming concepts and of the R language. However, some of these tools|in particular the statistical graphics and novel analytical methods|are also useful for nonprogrammers. To this end, we have developed an open source, extensible graphical user interface (GUI iFlow, which sits on top of the Bioconductor backbone, enabling basic analyses by means of convenient graphical menus and wizards. We envision iFlow to be easily extensible in order to quickly integrate novel methodological developments.

  20. Ultrasonic analyte concentration and application in flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2015-07-07

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  1. Ultrasonic analyte concentration and application in flow cytometry

    Energy Technology Data Exchange (ETDEWEB)

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2014-07-22

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  2. Ultrasonic analyte concentration and application in flow cytometry

    Science.gov (United States)

    Kaduchak, Gregory; Goddard, Greg; Salzman, Gary; Sinha, Dipen; Martin, John C.; Kwiatkowski, Christopher; Graves, Steven

    2008-03-11

    The present invention includes an apparatus and corresponding method for concentrating analytes within a fluid flowing through a tube using acoustic radiation pressure. The apparatus includes a function generator that outputs a radio frequency electrical signal to a transducer that transforms the radio frequency electric signal to an acoustic signal and couples the acoustic signal to the tube. The acoustic signal is converted within the tube to acoustic pressure that concentrates the analytes within the fluid.

  3. National flow cytometry and sorting research resource. Annual progress report, July, 1, 1994--June 30, 1995, Year 12

    Energy Technology Data Exchange (ETDEWEB)

    Jett, J.H.

    1995-04-27

    Research progress utilizing flow cytometry is described. Topics include: rapid kinetics flow cytometry; characterization of size determinations for small DNA fragments; statistical analysis; energy transfer measurements of molecular confirmation in micelles; and enrichment of Mus spretus chromosomes by dual parameter flow sorting and identification of sorted fractions by fluorescence in-situ hybridization onto G-banded mouse metaphase spreads.

  4. Flow Cytometry Sorting to Separate Viable Giant Viruses from Amoeba Co-culture Supernatants

    Science.gov (United States)

    Khalil, Jacques Y. B.; Langlois, Thierry; Andreani, Julien; Sorraing, Jean-Marc; Raoult, Didier; Camoin, Laurence; La Scola, Bernard

    2017-01-01

    Flow cytometry has contributed to virology but has faced many drawbacks concerning detection limits, due to the small size of viral particles. Nonetheless, giant viruses changed many concepts in the world of viruses, as a result of their size and hence opened up the possibility of using flow cytometry to study them. Recently, we developed a high throughput isolation of viruses using flow cytometry and protozoa co-culture. Consequently, isolating a viral mixture in the same sample became more common. Nevertheless, when one virus multiplies faster than others in the mixture, it is impossible to obtain a pure culture of the minority population. Here, we describe a robust sorting system, which can separate viable giant virus mixtures from supernatants. We tested three flow cytometry sorters by sorting artificial mixtures. Purity control was assessed by electron microscopy and molecular biology. As proof of concept, we applied the sorting system to a co-culture supernatant taken from a sample containing a viral mixture that we couldn't separate using end point dilution. In addition to isolating the quick-growing Mimivirus, we sorted and re-cultured a new, slow-growing virus, which we named “Cedratvirus.” The sorting assay presented in this paper is a powerful and versatile tool for separating viral populations from amoeba co-cultures and adding value to the new field of flow virometry. PMID:28111619

  5. Cell-cycle analysis of fission yeast cells by flow cytometry.

    Directory of Open Access Journals (Sweden)

    Jon Halvor Jonsrud Knutsen

    Full Text Available The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1 and G(2 phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1 phase. We have devised a flow cytometric method exploiting the fact that cells in G(1 phase contain two nuclei, whereas cells in G(2 are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1 and in G(2 phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

  6. Cell-cycle analysis of fission yeast cells by flow cytometry.

    Science.gov (United States)

    Knutsen, Jon Halvor Jonsrud; Rein, Idun Dale; Rothe, Christiane; Stokke, Trond; Grallert, Beáta; Boye, Erik

    2011-02-28

    The cell cycle of the fission yeast, Schizosaccharomyces pombe, does not easily lend itself to analysis by flow cytometry, mainly because cells in G(1) and G(2) phase contain the same amount of DNA. This occurs because fission yeast cells under standard growth conditions do not complete cytokinesis until after G(1) phase. We have devised a flow cytometric method exploiting the fact that cells in G(1) phase contain two nuclei, whereas cells in G(2) are mononuclear. Measurements of the width as well as the total area of the DNA-associated fluorescence signal allows the discrimination between cells in G(1) and in G(2) phase and the cell-cycle progression of fission yeast can be followed in detail by flow cytometry. Furthermore, we show how this method can be used to monitor the timing of cell entry into anaphase. Fission yeast cells tend to form multimers, which represents another problem of flow cytometry-based cell-cycle analysis. Here we present a method employing light-scatter measurements to enable the exclusion of cell doublets, thereby further improving the analysis of fission yeast cells by flow cytometry.

  7. Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

    Institute of Scientific and Technical Information of China (English)

    Md.Sharoare Hossain; Anders Johannisson; Margareta Wallgren; Szabolcs Nagy; Amanda Pimenta Siqueira; Heriberto Rodriguez-Martinez

    2011-01-01

    Flow cytometry is now a recognized methodology within animal spermatology, and has moved from being a research tool to become routine in the assessment of animal semen destined to breeding. The availability of 'bench-top' flow cytometers and of newer and versatile markers for cell structure and function had allowed the instrumentation to measure more sperm parameters, from viability to reactiveness when exposed to exogenous stimuli, and to increase our capabilities to sort spermatozoa for potential fertilizing capacity, or chromosomal sex. The present review summarizes the state of the art regarding flow cytometry applied to animal andrology, albeit keeping an open comparative intent. It critically evaluates the present and future capabilities of flow cytometry for the diagnostics of potential fertility and for the development of current reproductive technologies such as sperm freezing, sperm selection and sperm sorting. The flow cytometry methods will probably further revolutionize our understanding of the sperm physiology and their functionality, and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa. However, continuous follow-up of the methods is a necessity owing to technical developments and the complexity of mapping spermatozoa.

  8. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor

    Science.gov (United States)

    Cedeño-Arias, Mercedes; Sánchez-Ramírez, Javier; Blanco-Santana, Rancés; Rengifo-Calzado, Enrique

    2011-01-01

    An ideal test used to characterize a product must be appropriate for the measurement of product quality, manufacturing consistency, product stability, and comparability studies. Flow cytometry has been successfully applied to the examination of antibodies and receptors on membrane surfaces; however, to date, the analytical validation of cytometry based assays is limited. Here we report on the validation of a flow cytometry-based assay used in the evaluation of nimotuzumab binding to cells over-expressing EGFR on cell surface. The assay was validated by examining, assay robustness, specificity, repeatability and intermediate precision. The assay was highly specific, robust for all studied factors except for cell fixation with 1% paraformaldehyde and met criteria for precision with RSD < 2%. In addition the assay has stability-indicating properties evidenced by the ability to detect changes in mAb degraded samples. Most importantly, the assay demonstrated to be useful for its intended use. PMID:21886904

  9. Sex-sorting sperm using flow cytometry/cell sorting.

    Science.gov (United States)

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    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. Flow cytometry-based DNA hybridization and polymorphism analysis

    Energy Technology Data Exchange (ETDEWEB)

    Cai, H.; Kommander, K.; White, P.S.; Nolan, J.P.

    1998-07-01

    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.

  11. Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM).

    Science.gov (United States)

    Tang, Anson H L; Lai, Queenie T K; Chung, Bob M F; Lee, Kelvin C M; Mok, Aaron T Y; Yip, G K; Shum, Anderson H C; Wong, Kenneth K Y; Tsia, Kevin K

    2017-06-28

    Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture - information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization

  12. Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data.

    Science.gov (United States)

    Weber, Lukas M; Robinson, Mark D

    2016-12-01

    Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets. © 2016 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

  13. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid...

  14. Data File Standard for Flow Cytometry, Version FCS 3.1

    Energy Technology Data Exchange (ETDEWEB)

    Spidlen, Josef; Moore, Wayne; Parks, David; Goldberg, Michael; Bray, Chris; Gorombey, Peter; Hyun, Bill; Hubbard, Mark; Lange, Simon; Lefebvre, Ray; Leif, Robert; Novo, David; Ostruszka, Leo; Treister, Adam; Wood, James; Murphy, Robert F.; Roederer, Mario; Sudar, Damir; Zigon, Robert; Brinkman, Ryan R.; Brierre, Pierre

    2009-11-10

    The flow cytometry data file standard provides the specifications needed to completely describe flow cytometry data sets within the confines of the file containing the experimental data. In 1984, the first Flow Cytometry Standard format for data files was adopted as FCS 1.0. This standard was modified in 1990 as FCS 2.0 and again in 1997 as FCS 3.0. We report here on the next generation flow cytometry standard data file format. FCS 3.1 is a minor revision based on suggested improvements from the community. The unchanged goal of the standard is to provide a uniform file format that allows files created by one type of acquisition hardware and software to be analyzed by any other type. The FCS 3.1 standard retains the basic FCS file structure and most features of previous versions of the standard. Changes included in FCS 3.1 address potential ambiguities in the previous versions and provide a more robust standard. The major changes include simplified support for international characters and improved support for storing compensation. The major additions are support for preferred display scale, a standardized way of capturing the sample volume, information about originality of the data file, and support for plate and well identification in high throughput, plate based experiments. Please see the normative version of the FCS 3.1 specification in Supporting Information for this manuscript (or at http://www.isac-net.org/ in the Current standards section) for a complete list of changes.

  15. Plasmodium species: Flow cytometry and microfluorometry assessments of DNA content and synthesis

    NARCIS (Netherlands)

    Janse, C.J.; Vianen, P.H. van; Tanke, H.J.; Mons, B.; Ponnudurai, T.; Overdulve, J.P.

    1987-01-01

    Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were pe

  16. Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

    Science.gov (United States)

    Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

    2010-01-01

    The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to…

  17. High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

    DEFF Research Database (Denmark)

    Larsen, Jacob; Christensen, Knud; Larsen, Jørgen K;

    2004-01-01

    The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high......-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV...

  18. 78 FR 5186 - Clinical Flow Cytometry in Hematologic Malignancies; Public Workshop; Request for Comments

    Science.gov (United States)

    2013-01-24

    ... location. Webcast participants will be sent technical system requirements after registration and will be... need for such products to assist clinical laboratories in performing this testing. FDA has been working... HUMAN SERVICES Food and Drug Administration Clinical Flow Cytometry in Hematologic Malignancies; Public...

  19. Multiparametric flow cytometry profiling of neoplastic plasma cells in multiple myeloma

    DEFF Research Database (Denmark)

    Johnsen, Hans E; Bøgsted, Martin; Klausen, Tobias W;

    2010-01-01

    The clinical impact of multiparametric flow cytometry (MFC) in multiple myeloma (MM) is still unclear and under evaluation. Further progress relies on multiparametric profiling of the neoplastic plasma cell (PC) compartment to provide an accurate image of the stage of differentiation. The primary...

  20. DNA Aneuploidy by Flow Cytometry Is an Independent Prognostic Factor in Gastric Cancer

    Directory of Open Access Journals (Sweden)

    Mar Abad

    1998-01-01

    Full Text Available In the present study the prognostic value of both DNA ploidy and the proliferative activity of tomour cells were studied in a series of 76 consecutive patients suffering from gastric tumours. DNA ploidy and the proliferative index (as measured by the percentage of S-phase cells were determined by flow cytometry using fresh tumour specimens.

  1. A liposome-based size calibration method for measuring microvesicles by flow cytometry

    DEFF Research Database (Denmark)

    Simonsen, Jens Bæk

    2016-01-01

    BACKGROUND: Over the last years the need for a gold standard to determine the sizes of extracellular vesicles including microvesicles by flow cytometry has been emphasized. METHODS: This work suggests to use artificial vesicles as calibrators to ascertain the size of microvesicles from the side...

  2. DIRECT FLOW-CYTOMETRY OF ANAEROBIC-BACTERIA IN HUMAN FECES

    NARCIS (Netherlands)

    VANDERWAAIJ, LA; MESANDER, G; LIMBURG, PC; VANDERWAAIJ, D

    1994-01-01

    We describe a flow cytometry method for analysis of noncultured anaerobic bacteria present in human fecal suspensions. Nonbacterial fecal compounds, bacterial fragments, and large aggregates could be discriminated from bacteria by staining with propidium iodide (PI) and setting a discriminator on PI

  3. Improved method for bacterial cell capture after flow cytometry cell sorting.

    Science.gov (United States)

    Guillebault, D; Laghdass, M; Catala, P; Obernosterer, I; Lebaron, P

    2010-11-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

  4. High resolution DNA flow cytometry of boar sperm cells in identification of boars carrying cytogenetic aberrations

    DEFF Research Database (Denmark)

    Larsen, Jacob; Christensen, Knud; Larsen, Jørgen K

    2004-01-01

    The cytogenetic quality of boars used for breeding determines the litter outcome and thus has large economical consequences. Traditionally, quality controls based on the examination of simple karyograms are time consuming and sometimes give uncertain results. As an alternative, the use of high-re......-resolution DNA flow cytometry on DAPI-stained sperm cell nuclei (CV...

  5. Immunophenotyping of chronic B-cell neoplasms: flow cytometry versus immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Afaf Abdel-Aziz Abdel-Ghafar

    2012-02-01

    Full Text Available Morphological differentiation between benign and malignant lymphoproliferative disorders (LPDs can be challenging. Immunophenotyping (IPT by either technique, flow cytometry or immunohistochemistry (IHC, is an important step in solving such difficulty. Thirty-five newly diagnosed patients with chronic B-cell neoplasms (11 chronic lymphocytic leukemia, 22 non Hodgkin lymphoma and 2 hairy cell leukemia were included in this study with age range from 20 to 70 years. Monoclonal antibodies surface expression using lymphoproliferative disorders panel (CD45, CD19, CD5, CD10, CD11c, CD20, CD22, CD23, CD38, CD79b, FMC7, CD103, CD25, kappa and lambda light chains by flow cytometry was done on bone marrow samples. CD20, CD5, CD23, Bcl-2, Bcl-6, kappa and lambda light chain immunostaining were performed on fixed bone marrow trephine biopsy specimen. The sensitivity of IHC was 81.8% in chronic lymphocytic leukemia (CLL and 100% in non Hodgkin lymphoma (NHL as regards CD20, 100% in both groups as regards CD5, 46% in CLL and 66.7% in NHL as regards CD23, 33.3% in CLL and 50% in NHL as regards kappa chain, 20% in CLL and 33.3% in NHL as regards lambda chain. We found that IHC and flow cytometry are equally effective in diagnosing CLL; however, IHC might be slightly more sensitive than flow cytometry in detecting bone marrow infiltration in NHL and hairy cell leukemia (HCL.

  6. Fluorescence Assisted Selection of Transformants (FAST): Using flow cytometry to select fungal transformants

    NARCIS (Netherlands)

    Vlaardingerbroek, I.; Beerens, B.; Shahi, S.; Rep, M.

    2015-01-01

    The availability of drug resistance markers for fungal transformation is often a limiting factor in both fungal genetics research and industrial applications. We describe a new technique using flow cytometry to select fungal transformants using well-known fluorescent proteins as markers for transfor

  7. Teaching the Microbial Growth Curve Concept Using Microalgal Cultures and Flow Cytometry

    Science.gov (United States)

    Forget, Nathalie; Belzile, Claude; Rioux, Pierre; Nozais, Christian

    2010-01-01

    The microbial growth curve is widely studied within microbiology classes and bacteria are usually the microbial model used. Here, we describe a novel laboratory protocol involving flow cytometry to assess the growth dynamics of the unicellular microalgae "Isochrysis galbana." The algal model represents an appropriate alternative to bacteria…

  8. Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways**

    Science.gov (United States)

    Rationale: The evaluation of sputum leukocytes by flow cytometry is an opportunity to assess characteristics of cells residing in the central airways, yet it is hampered by certain inherent properties of sputum including mucus and large amounts of contaminating cells and debris. ...

  9. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders

    DEFF Research Database (Denmark)

    Rawstron, A.C.; Orfao, A.; Beksac, M.

    2008-01-01

    . A preferred panel would also include CD20, CD117, CD28 and CD27; 4) discrepancies between the percentage of plasma cells detected by flow cytometry and morphology are primarily related to sample quality and it is, therefore, important to determine that marrow elements are present in follow-up samples...

  10. External quality assessment in flow cytometry: educational aspects and trends toward improvement

    NARCIS (Netherlands)

    W.H.B.M. Levering

    2007-01-01

    textabstractFlow cytometry (FCM) uses the principles of hydro- dynamic focusing, light scattering, light excitation, and emission of fluorochrome molecules to generate specific multi-parameter data from particles and cells. FCM became rapidly a routine method for clinical decision-making in

  11. Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry

    DEFF Research Database (Denmark)

    Shitu, J. O.; Woodley, John; Wnek, R.

    2009-01-01

    The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O-2 uptake rate, CO2 evolution rate and optical density measurements). Induction...

  12. Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

    Science.gov (United States)

    Sinigalliano, Christopher D; Winshell, Jamie; Guerrero, Maria A; Scorzetti, Gloria; Fell, Jack W; Eaton, Richard W; Brand, Larry; Rein, Kathleen S

    2009-07-01

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

  13. Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.

    Science.gov (United States)

    Bendix, M; Dige, A; Deleuran, B; Dahlerup, J F; Jørgensen, S P; Bartels, L E; Husted, L B; Harsløf, T; Langdahl, B; Agnholt, J

    2015-07-01

    Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.

  14. Silica Microspheres Are Superior to Polystyrene for Microvesicle Analysis by Flow Cytometry

    Science.gov (United States)

    2015-02-16

    Regular Article Silica microspheres are superior to polystyrene for microvesicle analysis by flow cytometry☆ Bijaya Kumar Parida ⁎, Hiram Garrastazu...February 2015 Available online 16 February 2015 Keywords: Microvesicles Cell-derived microparticles Silica microspheres Polystyrene microspheres ...is used to characterize MVs. Polystyrene microspheres are often used in flow cytometry to distinguish MV from cells by setting a 1-μm MV gate in a

  15. Overview of the New Flow Cytometry RG and Proposed Cell Sorting (FACS) Microarray study

    OpenAIRE

    DeLay, Monica; Lopez, Peter; Tighe, Scott

    2013-01-01

    The Flow Cytometry Research Group (FCRG) is the latest addition to the ABRF RG family. The RG is currently in its first year and has 9 members; many of whom are flow cytometrists new to the ABRF. The initial goal of the FCRG is to describe a method for the evaluation of cell stress or other deleterious perturbations caused by cell sorting across a wide range of cell types.

  16. Overview of the New Flow Cytometry RG and Proposed Cell Sorting (FACS) Microarray study

    OpenAIRE

    2013-01-01

    The Flow Cytometry Research Group (FCRG) is the latest addition to the ABRF RG family. The RG is currently in its first year and has 9 members; many of whom are flow cytometrists new to the ABRF. The initial goal of the FCRG is to describe a method for the evaluation of cell stress or other deleterious perturbations caused by cell sorting across a wide range of cell types.

  17. Flow cytometry analysis of FITC-labeled concanavalin A binding to human blood cells as an indicator of radiation-induced membrane alterations

    Energy Technology Data Exchange (ETDEWEB)

    Donnadieu-Claraz, M.; Paillole, N.; Voisin, P. [CEA Centre d`Etudes de Fontenay-aux-Roses, 92 (France). Dept. de Protection de la Sante de l`Homme et de Dosimetrie; Djounova, J. [National Centre of Radiobiology and Radiation Protection, Sofia (Bulgaria)

    1995-12-31

    The {sup 3}H concanavalin-A binding to human blood cells have been described as a promising biological indicator of radiation overexposure. Flow cytometry adaptation of this technique using fluorescein-labelled concanavalin-A were performed to estimate time-dependent changes in binding on human blood cells membranes after in vitro {gamma} irradiation ({sup 60}Co). Result revealed significant enhanced lectin-binding to platelets and erythrocytes in a dose range of 0,5-5 Gy, 1 and 3 hours after irradiation. However for both platelets and erythrocytes, it was impossible to discriminate between the different doses. Further studies are necessary to confirm the suitability of lectin-binding as a biological indicator for radiation dose assessment. (authors). 5 refs., 1 fig.

  18. The relationship between fractional flow reserve, platelet reactivity and platelet leukocyte complexes in stable coronary artery disease.

    Directory of Open Access Journals (Sweden)

    Jan-Willem E M Sels

    Full Text Available BACKGROUND: The presence of stenoses that significantly impair blood flow and cause myocardial ischemia negatively affects prognosis of patients with stable coronary artery disease. Altered platelet reactivity has been associated with impaired prognosis of stable coronary artery disease. Platelets are activated and form complexes with leukocytes in response to microshear gradients caused by friction forces on the arterial wall or flow separation. We hypothesized that the presence of significantly flow-limiting stenoses is associated with altered platelet reactivity and formation of platelet-leukocyte complexes. METHODS: One hundred patients with stable angina were studied. Hemodynamic significance of all coronary stenoses was assessed with Fractional Flow Reserve (FFR. Patients were classified FFR-positive (at least one lesion with FFR≤0.75 or FFR-negative (all lesions FFR>0.80. Whole blood samples were stimulated with increasing concentrations of ADP, TRAP, CRP and Iloprost with substimulatory ADP. Expression of P-selectin as platelet activation marker and platelet-leukocyte complexes were measured by flowcytometry. Patients were stratified on clopidogrel use. FFR positive and negative patient groups were compared on platelet reactivity and platelet-leukocyte complexes. RESULTS: Platelet reactivity between FFR-positive patients and FFR-negative patients did not differ. A significantly lower percentage of circulating platelet-neutrophil complexes in FFR-positive patients and a similar non-significant decrease in percentage of circulating platelet-monocyte complexes in FFR-positive patients was observed. CONCLUSION: The presence of hemodynamically significant coronary stenoses does not alter platelet reactivity but is associated with reduced platelet-neutrophil complexes in peripheral blood of patients with stable coronary artery disease.

  19. Non-Linear Optical Flow Cytometry Using a Scanned, Bessel Beam Light-Sheet

    Science.gov (United States)

    Collier, Bradley B.; Awasthi, Samir; Lieu, Deborah K.; Chan, James W.

    2015-01-01

    Modern flow cytometry instruments have become vital tools for high-throughput analysis of single cells. However, as issues with the cellular labeling techniques often used in flow cytometry have become more of a concern, the development of label-free modalities for cellular analysis is increasingly desired. Non-linear optical phenomena (NLO) are of growing interest for label-free analysis because of the ability to measure the intrinsic optical response of biomolecules found in cells. We demonstrate that a light-sheet consisting of a scanned Bessel beam is an optimal excitation geometry for efficiently generating NLO signals in a microfluidic environment. The balance of photon density and cross-sectional area provided by the light-sheet allowed significantly larger two-photon fluorescence intensities to be measured in a model polystyrene microparticle system compared to measurements made using other excitation focal geometries, including a relaxed Gaussian excitation beam often used in conventional flow cytometers. PMID:26021750

  20. Two-step protocol for preparing adherent cells for high-throughput flow cytometry.

    Science.gov (United States)

    Kaur, Mandeep; Esau, Luke

    2015-09-01

    We have developed a simple, cost-effective, and labor-efficient two-step protocol for preparing adherent cells for high-throughput flow cytometry. Adherent cells were grown on microplates, detached with 2.9 mM EDTA (pH 6.14) added directly to wells containing cell culture medium, stained, and then analyzed on a flow cytometer. This protocol bypasses washing, centrifugation, and transfer between plates, reducing the cell loss that occurs in standard multistep protocols. The method has been validated using six adherent cell lines, four commercially available dyes, and two antibodies; the results have been confirmed using two different flow cytometry (FC) instruments. Our approach has been used for estimating apoptosis, mitochondrial membrane potential, reactive oxygen species, and autophagy in response to exposure to pure compounds as well as plant and bacterial extracts.

  1. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

    Directory of Open Access Journals (Sweden)

    Josep M. Gasol

    2000-06-01

    Full Text Available Flow cytometry is rapidly becoming a routine methodology in aquatic microbial ecology. The combination of simple to use bench-top flow cytometers and highly fluorescent nucleic acid stains allows fast and easy determination of microbe abundance in the plankton of lakes and oceans. The different dyes and protocols used to stain and count planktonic bacteria as well as the equipment in use are reviewed, with special attention to some of the problems encountered in daily routine practice such as fixation, staining and absolute counting. One of the main advantages of flow cytometry over epifluorescence microscopy is the ability to obtain cell-specific measurements in large numbers of cells with limited effort. We discuss how this characteristic has been used for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content, and for estimating the relative activity and physiological state of each cell. We also describe how some of the flow cytometrically obtained data can be used to characterize the role of microbes on carbon cycling in the aquatic environment and we prospect the likely avenues of progress in the study of planktonic prokaryotes through the use of flow cytometry.

  2. Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

    Science.gov (United States)

    Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

    2016-09-01

    Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

  3. Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

    Institute of Scientific and Technical Information of China (English)

    Ekaterina I Galanzha; Vladimir P Zharov; Philips Classic

    2007-01-01

    Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo.Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry,super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., realtime quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers,drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and highspeed imaging of cell transient deformability in flow.In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell's functional state (e.g., live, apoptotic, or necrotic),and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

  4. FLOW CYTOMETRY AS A MODERN ANALYTICAL TOOL IN BIOLOGY AND MEDICINE

    Directory of Open Access Journals (Sweden)

    S. V. Khaidukov

    2007-01-01

    Full Text Available Abstract. Flow cytometry is considered as a modern technology for fast measurements of cellular characteristics, their organelles, and processes occurring within them. It is regarded as an efficient solution in many important areas of cell biology, immunology and cellular engineering. Present article bears on main developments in flow cytometry and their applications in medical and biological practice. Usage of modern achievements in fluorescent dyes, progress in laser and computer technologies, as well as potent software, resulted in wide application of this technique in medical practice. Accordingly, usage of monoclonal antibodies conjugated to different fluorochromes has led to elaboration of multiparametric analysis and did sufficiently simplify specialized works aimed for diagnostics of various immune disorders. The new directions in flow cytometry, e.g., flow cytoenzymology, provide wide opportunities for detailed identification of damaged or altered cells, and taking adequate decisions in treatment of detected pathological changes. The authors suggest that this article could initiate a series of publications concerning usage of this technology and its modern applications in broad laboratory practice.

  5. In Vivo Monitoring of Multiple Circulating Cell Populations Using Two-photon Flow Cytometry.

    Science.gov (United States)

    Tkaczyk, Eric R; Zhong, Cheng Frank; Ye, Jing Yong; Myc, Andrzej; Thomas, Thommey; Cao, Zhengyi; Duran-Struuck, Raimon; Luker, Kathryn E; Luker, Gary D; Norris, Theodore B; Baker, James R

    2008-02-15

    To detect and quantify multiple distinct populations of cells circulating simultaneously in the blood of living animals, we developed a novel optical system for two-channel, two-photon flow cytometry in vivo. We used this system to investigate the circulation dynamics in live animals of breast cancer cells with low (MCF-7) and high (MDA-MB-435) metastatic potential, showing for the first time that two different populations of circulating cells can be quantified simultaneously in the vasculature of a single live mouse. We also non-invasively monitored a population of labeled, circulating red blood cells for more than two weeks, demonstrating that this technique can also quantify the dynamics of abundant cells in the vascular system for prolonged periods of time. These data are the first in vivo application of multichannel flow cytometry utilizing two-photon excitation, which will greatly enhance our capability to study circulating cells in cancer and other disease processes.

  6. Automatic Clustering of Flow Cytometry Data with Density-Based Merging

    Directory of Open Access Journals (Sweden)

    Guenther Walther

    2009-01-01

    made this technology ubiquitous and indispensable in the clinical and laboratory setting. A current limit to the potential of this technology is the lack of automated tools for analyzing the resulting data. We describe methodology and software to automatically identify cell populations in flow cytometry data. Our approach advances the paradigm of manually gating sequential two-dimensional projections of the data to a procedure that automatically produces gates based on statistical theory. Our approach is nonparametric and can reproduce nonconvex subpopulations that are known to occur in flow cytometry samples, but which cannot be produced with current parametric model-based approaches. We illustrate the methodology with a sample of mouse spleen and peritoneal cavity cells.

  7. Analysis of polyethylene glycol (PEG) fusion in cultured neuroblastoma cells via flow cytometry: Techniques & optimization.

    Science.gov (United States)

    Hoffman, Ashley N; Bamba, Ravinder; Pollins, Alonda C; Thayer, Wesley P

    2017-02-01

    Polyethylene glycol (PEG) has long been used as a membrane fusogen, but recently it has been adopted as a technique for peripheral nerve repair. Vertebrate models using PEG fusion have shown improved outcomes when PEG is applied during repair of severed peripheral nerves. The cellular mechanism of PEG fusion in the peripheral nerve repair model has not previously been assessed via flow cytometry. PEG fusion was assessed in this experiment by dying B35 rat neuroblastoma cells with different color fluorescent labels. The different color cells were combined and PEG was applied in concentrations of 50%, 75% and 100%. The amount of cell fusion was assessed via flow cytometry as the percentage of double positive cells. Results showed increasing fusion and decreasing viability with increasing concentrations of PEG.

  8. Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Pick Neora

    2004-01-01

    Full Text Available The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

  9. Validation and quality control of hematolymphoid neoplasm immunophenotyping by flow cytometry

    Institute of Scientific and Technical Information of China (English)

    Wu Lijuan; Xu Dongsheng

    2012-01-01

    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.

  10. Application of flow cytometry and cell sorting to the bacterial analysis of environmental aerosol samples.

    Science.gov (United States)

    Hernlem, Bradley J; Ravva, Subbarao V

    2007-12-01

    Flow cytometry (FCM) combined with viability staining is a useful tool in discerning viable bacteria in environmental samples where traditional culture methods may fail. Contamination of aerosol samples with dust and other non-biological particles can interfere with accurate sample analysis and therefore there is a desire to exclude those particles from analysis. Particles were sorted according to their light scattering properties, cultured and isolates obtained. Isolates were cultured in suspension and reanalyzed by flow cytometry. The isolates were also analyzed and identified by DNA sequence analysis. Isolates with statistically similar light scattering properties shared common sequence identification. Isolates exhibited distinct light scattering profiles that roughly correlated with their originating gate, but often the peak of the profile was outside that gate.

  11. Use of Flow Cytometry to Measure Biogeochemical Rates and Processes in the Ocean

    Science.gov (United States)

    Lomas, Michael W.; Bronk, Deborah A.; van den Engh, Ger

    2011-01-01

    An important goal of marine biogeochemists is to quantify the rates at which elements cycle through the ocean's diverse microbial assemblage, as well as to determine how these rates vary in time and space. The traditional view that phytoplankton are producers and bacteria are consumers has been found to be overly simplistic, and environmental metagenomics is discovering new and important microbial metabolisms at an accelerating rate. Many nutritional strategies previously attributed to one microorganism or functional group are also or instead carried out by other groups. To tease apart which organism is doing what will require new analytical approaches. Flow cytometry, when combined with other techniques, has great potential for expanding our understanding of microbial interactions because groups can be distinguished optically, sorted, and then collected for subsequent analyses. Herein, we review the advances in our understanding of marine biogeochemistry that have arisen from the use of flow cytometry.

  12. OPPORTUNITIES OF FLOW CYTOMETRY IN DIAGNOSTICS OF INFECTIOUS DISEASES. PART 1

    Directory of Open Access Journals (Sweden)

    S. V. Khaidukov

    2011-01-01

    Full Text Available Abstract. Development of a modern medical science demands more thorough and deeper approach to diagnostics of diseases. New diagnostic technologies for these investigations are necessary and flow cytometry is such technology. Modern achievements in the field of fluorescent dyes, development of laser and computer technologies, have led to wide use of the given technology in medical practice. Infectious immunology is not exception. Cytokines production analysis at tuberculosis, HLA-DR expression changes for monocytes and CD64 for granulocytes at sepsis are the important diagnostic attributes. Flow cytometry offers new approaches to early and faster diagnostics of infringements of immune system at tuberculosis and sepsis, allows supervising development of these pathologies more effectively and estimate efficiency of therapy.

  13. OPPORTUNITIES OF FLOW CYTOMETRY IN DIAGNOSTICS OF INFECTIOUS DISEASES. Part 2

    Directory of Open Access Journals (Sweden)

    S. V. Khaidukov

    2011-01-01

    Full Text Available Abstract. Flow cytometry allows estimating quantitative and qualitative structure of populations and subpopulations of immune system cells by using various methodical approaches and a wide spectrum of reagents. For diagnostics the Acquired Immune Deficiency Syndrome (AIDS caused by a Human Immunodeficiency Virus (HIV the flow cytometry became irreplaceable. Traditionally, immunologists examine standard model of an estimation of immune dysfunction on the basis of classical markers of Т-cells (CD3, CD4, CD8 at the HIV-infection. But researchers pay less attention to other populations and subpopulations of lymphocytes, such as γδ-, αβ- and CD38+ Т-cells. The quantitative estimation of these parameters from a HIV and AIDS patients enables to see pathogenesis a HIV infection and the prediction of its development from another side. 

  14. Detection of Intracellular Factor VIII Protein in Peripheral Blood Mononuclear Cells by Flow Cytometry

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    Gouri Shankar Pandey

    2013-01-01

    Full Text Available Flow cytometry is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. Detection of specific host proteins for diagnosis predominantly uses quantitative PCR and western blotting assays. In this study, we optimized a flow cytometry-based detection assay for Factor VIII protein in peripheral blood mononuclear cells (PBMCs. An indirect intracellular staining (ICS method was standardized using monoclonal antibodies to different domains of human Factor VIII protein. The FVIII protein expression level was estimated by calculating the mean and median fluorescence intensities (MFI values for each monoclonal antibody. ICS staining of transiently transfected cell lines supported the method's specificity. Intracellular FVIII protein expression was also detected by the monoclonal antibodies used in the study in PBMCs of five blood donors. In summary, our data suggest that intracellular FVIII detection in PBMCs of hemophilia A patients can be a rapid and reliable method to detect intracellular FVIII levels.

  15. Validation of algal viability treated with total residual oxidant and organic matter by flow cytometry.

    Science.gov (United States)

    Lee, Junghyun; Choi, Eun Joo; Rhie, Kitae

    2015-08-15

    Algal cell growth after starch and oxidant treatments in seawater species (Isochrysis galbana and Phaeodactylum tricornutum) and freshwater species (Selenastrum capricornutum and Scenedesmus obliquus) were evaluated by flow cytometry with fluorescein diacetate (FDA) staining to determine algal viability. Growth of algal cell was found to be significantly different among groups treated with NaOCl, starch and/or sodium thiosulfate, which are active substance (Total Residual Oxidant; TRO as Cl2), organic compound to meet efficacy testing standard and neutralizer of TRO by Ballast Water Management Convention of International Maritime Organization, respectively. The viability of algal cell treated with TRO in starch-add culture of 5days after treatment and neutralization was decreased significantly. ATP contents of the treated algal cells corresponded to the FL1 fluorescent signal of flow cytometry with FDA staining. I. galbana was the most sensitive to TRO-neutralized cultures during viability analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Determination of total bacterial count in raw milk by flow cytometry

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    Dubravka Samaržija

    2004-01-01

    Full Text Available The automatic flow cytometry as routine method for total bacterial count determination of raw ex-farm milk has recently been accepted in Croatia. This method significantly differs from the reference method (Standard Plate Count mostly in the presentation of the results obtained. Therefore, this paper summarized experiences in the application of flow cytometry in the dairy laboratories practice. The principle and the practice of the method, methodological details and factors influencing the results were described. In order to avoid problems regarding the interpretation of the results, which aregeneral problems of the quantitative microbiology, this article try to explain an appropriate conversion of the results with regards to SPC/ml, as an official method for the bacteriological quality proposal by the national legislation.

  17. Application of flow cytometry in diagnosing lymphomas in dogs and cats.

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    Aniołek, Olga; Gajewski, Zdzisław; Giziński, Sławomir

    2014-01-01

    Classification of types of lymphomas is done by interpreting cell morphology results obtained in cytological and/or histological examinations. In recent years, additional methods like immunocytochemistry (ICC), immunohistochemistry (IHC), immunophenotyping by flow cytometry and polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR), have been used to diagnose and classify lymphomas. Unfortunately, none of these methods is completely specific and sensitive. Thus, a combination of several diagnostic methods or use of all available techniques allows for evaluation of morphological properties of cells like their maturity and diversification. Owing to the use of sets of antibodies it is possible to identify the phenotype of hyperplastic cells as well as their origin. Combination of results obtained through phenotypical analysis with flow cytometry examination with morphological, histological and genetic testing enables a detailed analysis of, in this case, lymphoproliferative diseases including reaction changes, primary and secondary immunological deficits as well as autoimmune diseases.

  18. An automated analysis of highly complex flow cytometry-based proteomic data.

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    Stuchlý, Jan; Kanderová, Veronika; Fišer, Karel; Cerná, Daniela; Holm, Anders; Wu, Weiwei; Hrušák, Ondřej; Lund-Johansen, Fridtjof; Kalina, Tomáš

    2012-02-01

    The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.

  19. Rapid parallel flow cytometry assays of active GTPases using effector beads.

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    Buranda, Tione; BasuRay, Soumik; Swanson, Scarlett; Agola, Jacob; Bondu, Virginie; Wandinger-Ness, Angela

    2013-11-15

    We describe a rapid assay for measuring the cellular activity of small guanine triphosphatases (GTPases) in response to a specific stimulus. Effector-functionalized beads are used to quantify in parallel multiple GTP-bound GTPases in the same cell lysate by flow cytometry. In a biologically relevant example, five different Ras family GTPases are shown for the first time to be involved in a concerted signaling cascade downstream of receptor ligation by Sin Nombre hantavirus.

  20. Integration of lyoplate based flow cytometry and computational analysis for standardized immunological biomarker discovery.

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    Federica Villanova

    Full Text Available Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid flow cytometry platform (CFP and a unique lyoplate-based flow cytometry platform (LFP in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10 and activation markers (Foxp3 and CD25. Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

  1. Application of flow cytometry in marine phytoplankton research: current applications and future perspectives

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    Marcel J.W. Veldhuis

    2000-06-01

    Full Text Available A brief overview is given of current applications of flow cytometry (FCM in marine phytoplankton research. This paper presents a selection of highlights and various technical and analytical problems we encountered during the past 10 years. In particular, the conversion of the relative values obtained in terms of size and fluorescence applying FCM to quantitative estimates of cell size, pigment concentration, genome size etc., is addressed. The introduction of DNA -cell-cycle analysis made easily assessable by flow cytometry has been of great importance, allowing in situ measurement of species specific growth rates. Key questions in ecology such as factors determining the wax and wane of phytoplankton bloom can now be better answered in terms of species specific growth and mortality. Finally, flow cytometry provides detailed information of the physiological status of the individual algal cells. New staining methods enable us to distinguish between viable and non-viable cells and so will help us to elucidate the importance of automortality in aquatic ecosystems.

  2. Discovering cell types in flow cytometry data with random matrix theory

    Science.gov (United States)

    Shen, Yang; Nussenblatt, Robert; Losert, Wolfgang

    Flow cytometry is a widely used experimental technique in immunology research. During the experiments, peripheral blood mononuclear cells (PBMC) from a single patient, labeled with multiple fluorescent stains that bind to different proteins, are illuminated by a laser. The intensity of each stain on a single cell is recorded and reflects the amount of protein expressed by that cell. The data analysis focuses on identifying specific cell types related to a disease. Different cell types can be identified by the type and amount of protein they express. To date, this has most often been done manually by labelling a protein as expressed or not while ignoring the amount of expression. Using a cross correlation matrix of stain intensities, which contains both information on the proteins expressed and their amount, has been largely ignored by researchers as it suffers from measurement noise. Here we present an algorithm to identify cell types in flow cytometry data which uses random matrix theory (RMT) to reduce noise in a cross correlation matrix. We demonstrate our method using a published flow cytometry data set. Compared with previous analysis techniques, we were able to rediscover relevant cell types in an automatic way. Department of Physics, University of Maryland, College Park, MD 20742.

  3. An assessment of software for flow cytometry analysis in banana plants

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    Renata Alves Lara Silva

    2014-02-01

    Full Text Available Flow cytometry is a technique that yields rapid results in analyses of cell properties such as volume, morphological complexity and quantitative DNA content, and it is considered more convenient than other techniques. However, the analysis usually generates histograms marked by variations that can be produced by many factors, including differences between the software packages that capture the data generated by the flow cytometer. The objective of the present work was to evaluate the performance of four software products commonly used in flow cytometry based on quantifications of DNA content and analyses of the coefficients of variation associated with the software outputs. Readings were obtained from 25 ‘NBA’ (AA banana leaf samples using the FACSCalibur (BD flow cytometer, and 25 histograms from each software product (CellQuest™, WinMDI™, FlowJo™ and FCS Express™ were analyzed to obtain the estimated DNA content and the coefficient of variation (CV of the estimates. The values of DNA content obtained from the software did not differ significantly. However, the CV analysis showed that the precision of the WinMDI™ software was low and that the CV values were underestimated, whereas the remaining software showed CV values that were in relatively close agreement with those found in the literature. The CellQuest™ software is recommended because it was developed by the same company that produces the flow cytometer used in the present study.

  4. Best practices in performing flow cytometry in a regulated environment: feedback from experience within the European Bioanalysis Forum.

    Science.gov (United States)

    der Strate, Barry van; Longdin, Robin; Geerlings, Marie; Bachmayer, Nora; Cavallin, Maria; Litwin, Virginia; Patel, Minesh; Passe-Coutrin, Wilfried; Schoelch, Corinna; Companjen, Arjen; Fjording, Marianne Scheel

    2017-08-01

    Flow cytometry is a powerful tool that can be used for the support of (pre)clinical studies. Although various white papers are available that describe the set-up and validation of the instrumentation (the flow cytometer) and validation of flow cytometry methods, to date no guidelines exist that address the requirements for performing flow cytometry in a regulated environment. In this manuscript, the European Bioanalysis Forum presents additional practice guidance on the use of flow cytometry in the support of drug development programs and addresses areas that are not covered in the previous publications. The concepts presented here are based on the consensus of discussions in the European Bioanalysis Forum Topic Team 32, in meetings in Barcelona, Limelette and multiple telephone conferences.

  5. Flow Cytometry Enables Multiplexed Measurements of Genetically Encoded Intramolecular FRET Sensors Suitable for Screening.

    Science.gov (United States)

    Doucette, Jaimee; Zhao, Ziyan; Geyer, Rory J; Barra, Melanie M; Balunas, Marcy J; Zweifach, Adam

    2016-07-01

    Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

  6. A high-throughput method for detection of DNA in chloroplasts using flow cytometry

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    Oldenburg Delene J

    2007-03-01

    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.

  7. Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls.

    Science.gov (United States)

    Christensen, Preben; Boelling, Dorothee; Pedersen, Kurt Myrup; Korsgaard, Inge Riis; Jensen, Just

    2005-01-01

    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.

  8. Platelets

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    ... tiny fraction of the blood volume. The principal function of platelets is to prevent bleeding. Red blood cells are ... forming a long string. This illustrates the basic function of platelets, to stick to any foreign surface and then ...

  9. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    Science.gov (United States)

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

    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.

  10. Evaluation of cell proliferative activity after irradiation using immunohistochemical approach and flow cytometry

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    Tamada, Takashi (Okayama Univ. (Japan). School of Medicine)

    1992-06-01

    To evaluate a proliferative activity of post-irradiated malignant cells, we studied the kinetics of HeLa cells using immunohistochemical approach and flow cytometry. HeLa cells were stained with two proliferation-associated monoclonal antibodies, Ki-67 and anti-DNA polymerase {alpha} antibody. Nucleoli of non-irradiated cells were granularly stained with Ki-67. After irradiation, only the center of nuclei was diffusely stained with Ki-67. One hundred forty-four hours after low-dose irradiation, the staining patterns became the same as the control. On the other hand, after high-dose irradiation, the center of nuclei was weakly stained. DNA polymerase {alpha} was diffusely labelled with nuclei of the control. It was located around the border of nuclei of low-dose irradiated cells like a ring. But after high-dose irradiation, it was granularly distributed in the periphery of nuclei. FITC conjugated Ki-67/PI two parameter analysis was done by a single laser flow cytometer. Twenty-four hours after irradiation, DNA-histograms showed the accumulation to G{sub 2}/M phase and the increase of DNA content of G{sub 2}/M cells, as exposure dose was increased. Two parameter analysis showed the increase of FITC uptake of G{sub 2}/M phase as dose increased. These changes of flow cytometry were remarkably observed after 24 hours' incubation. It was shown that the difference of Ki-67 antigen and DNA polymerase {alpha} appearance depended on the irradiation dose. These findings suggest that immunohistochemical staining with Ki-67 or anti-DNA polymerase {alpha} antibody and flow cytometry using Ki-67 are available to evaluate cell damages after irradiation. (author).

  11. Comparison of Flow Cytometry and ELASA for Screening of Proper Candidate Aptamer in Cell-SELEX Pool.

    Science.gov (United States)

    Nabavinia, Maryam Sadat; Charbgoo, Fahimeh; Alibolandi, Mona; Mosaffa, Fatemeh; Gholoobi, Aida; Ramezani, Mohammad; Abnous, Khalil

    2017-07-19

    Aptamers are single-stranded RNA or DNA, which bind to their target with high affinity and specificity. Method of isolating aptamers against cell surface protein is called cell-SELEX. Common approach for monitoring cell-SELEX developed aptamers is flow cytometry. Since flow cytometry is costly and requires sophisticated equipments, we suggested implementing easy access, high throughput enzyme-link apta-sorbent assay test (ELASA) to confirm the specificity of aptamers selected through cell-SELEX process. In this regard, we compared ELASA and flow cytometry techniques in order to screen potent candidate aptamers against A2780 Rcis cell line, which were selected by cell-SELEX. The obtained results demonstrated that both ELASA and flow cytometry are identical in terms of sensivity and precision for aptamers selection. Then it could be concluded that ELASA method could be used as a versatile, inexpensive procedure for in vito evaluation of isolated aptamers from cell-SELEX based process.

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

    Science.gov (United States)

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

    2016-02-01

    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.

  13. A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

    Science.gov (United States)

    Hendon-Dunn, Charlotte Louise; Doris, Kathryn Sarah; Thomas, Stephen Richard; Allnutt, Jonathan Charles; Marriott, Alice Ann Neville; Hatch, Kim Alexandra; Watson, Robert James; Bottley, Graham; Marsh, Philip David; Taylor, Stephen Charles; Bacon, Joanna

    2016-07-01

    Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

  14. Analysis of nuclear localization of interleukin-1 family cytokines by flow cytometry.

    Science.gov (United States)

    Ross, Ralf; Grimmel, Jan; Goedicke, Sybelle; Möbus, Anna M; Bulau, Ana-Maria; Bufler, Philip; Ali, Shafaqat; Martin, Michael U

    2013-01-31

    The dual function cytokines IL-1α, IL-33 and IL-37 are members of the IL-1 cytokine family. Besides of being able to bind to their cognate receptors on target cells, they can act intracellularly in the producing cell. All three are able to translocate to the nucleus and have been discussed to affect gene expression. In order to compare and quantitate nuclear translocation of these IL-1 family members we established a robust technique which enables to measure nuclear localization on a single cell level by flow cytometry. Vectors encoding fusion proteins of different IL-1 family members with enhanced green fluorescent protein were cloned and cell lines transiently transfected with these. Fluorescent fusion proteins in intact cells or in isolated nuclei were detected subsequently by fluorescence microscopy and flow cytometry, respectively. Depending on the cellular system, cells and nuclei were distinguishable by flow cytometry in forward scatter/sideward scatter. Fluorescent fusion proteins were detectable in isolated nuclei up to three days following preparation. Signal intensity of fusion proteins of IL-33 and IL-37 in isolated nuclei but not of IL-1α, was markedly increased by fixation with paraformaldehyde, directly following cell lysis, indicating that IL-1α binds stronger to nuclear structures than IL-33 and IL-37. Nuclear translocation of fluorescent IL-37 fusion proteins in a stably transfected RAW264.7 mouse macrophage cell line required stimulation with lipopolysaccharide. Applying this method we demonstrated that a prolonged lag phase of more than 15h before LPS-stimulated nuclear translocation was detected. In summary, we present a robust method to analyze and quantitate nuclear localization of IL-1 cytokine family members. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. High-throughput monitoring of plant nuclear DNA contents via flow cytometry.

    Science.gov (United States)

    Galbraith, David W; Lambert, Georgina M

    2012-01-01

    Interest in measuring the nuclear holoploid genome sizes of higher plants reflects not just the status of the nucleus as a defining characteristic of eukaryotic organisms. Higher plants also attract interest in that they display an unusually large range of genome sizes, current measurements indicating an almost 2,500-fold difference between the smallest and the largest. Scientists would like to learn more about the significance of nuclear genome sizes, in terms of molecular and cytological mechanisms regulating the interaction of the nucleus with the cytoplasm and regulating the observed increases and decreases of genome sizes observed within and across families, genera, and species. We would like to understand their adaptive significance through charting their distribution within populations and ecosystems. Further, since genome size values are only available for a small minority of the ∼650,000 species of angiosperms (known and yet undiscovered), we would like to systematically survey plant genome sizes globally before their extinction as a consequence of anthropogenic change. Flow cytometry is accepted as the method of choice for genome size measurements, these measurements being based on fluorescent staining of the nuclear DNA. Flow cytometry offers exceptional ease of use, accompanied by high accuracy and reproducibility and low cost. This chapter provides a general discussion of flow cytometric methods for measuring plant genome sizes, and detailed methods for carrying out these analyses.

  16. Novel quantitative autophagy analysis by organelle flow cytometry after cell sonication.

    Directory of Open Access Journals (Sweden)

    Michael Degtyarev

    Full Text Available Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication, takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis.

  17. Role of receptor occupancy assays by flow cytometry in drug development.

    Science.gov (United States)

    Stewart, Jennifer J; Green, Cherie L; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Wilkins, Danice E C; Moulard, Maxime; Czechowska, Kamila; Lanham, David; McCloskey, Thomas W; Ferbas, John; van der Strate, Barry W A; Högerkorp, Carl-Magnus; Wyant, Timothy; Lackey, Alan; Litwin, Virginia

    2016-03-01

    The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.

  18. In vivo flow cytometry and time-resolved near-IR angiography and lymphography

    Science.gov (United States)

    Galanzha, Ekaterina I.; Tuchin, Valery V.; Brock, Robert W.; Zharov, Vladimir P.

    2007-05-01

    Integration of photoacoustic and photothermal techniques with high-speed, high-resolution transmission and fluorescence microscopy shows great potential for in vivo flow cytometry and indocyanine green (ICG) near-infrared (IR) angiography of blood and lymph microvessels. In particular, the capabilities of in vivo flow cytometry using rat mesentery and nude mouse ear models are demonstrated for real-time quantitative detection of circulating and migrating individual blood and cancer cells in skin, mesentery, lymph nodes, liver, kidney; studying vascular dynamics with a focus on lymphatics; monitoring cell traffic between blood and lymph systems; high-speed imaging of cell deformability in flow; and label-free real-time monitoring of single cell extravasation from blood vessel lumen into tissue. As presented, the advantages of ICG IR-angiography include estimation of time resolved dye dynamics (appearance and clearance) in blood and lymph microvessels using fluorescent and photoacoustic modules of the integrated technique. These new approaches are important for monitoring and quantifying metastatic and apoptotic cells; comparative measurements of plasma and cell velocities; analysis of immune responses; monitoring of circulating macromolecules, chylomicrons, bacteria, viruses and nanoparticles; molecular imaging. In the future, we believe that the integrated technique presented will have great potential for translation to early disease diagnoses (e.g. cancer) or assessment of innovative therapeutic interventions in humans.

  19. Detection and capture of breast cancer cells with photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran; Goldschmidt, Benjamin S.; Viator, John A.

    2016-08-01

    According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis-the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems-significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  20. Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

    Directory of Open Access Journals (Sweden)

    Friedrich RP

    2015-06-01

    Full Text Available Ralf P Friedrich,1 Christina Janko,1 Marina Poettler,1 Philipp Tripal,1 Jan Zaloga,1 Iwona Cicha,1 Stephan Dürr,1,2 Johannes Nowak,3 Stefan Odenbach,3 Ioana Slabu,4 Maik Liebl,4 Lutz Trahms,4 Marcus Stapf,5 Ingrid Hilger,5 Stefan Lyer,1 Christoph Alexiou1 1Department of Otorhinolaryngology, Head and Neck Surgery, Section of Experimental Oncology and Nanomedicine, University hospital Erlangen, 2Department of Otorhinolaryngology, Head and Neck Surgery, Section of Phoniatrics and Pediatric Audiology, University hospital Erlangen, Erlangen, 3Technische Universität Dresden, Chair of Magnetofluiddynamics, Measuring and Automation Technology, Dresden, 4Physikalisch-Technische Bundesanstalt Berlin, Berlin, 5Department of Radiology, Division of Diagnostic and Interventional Radiology, Experimental Radiology, University hospital Jena, Jena, Germany Abstract: Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of superparamagnetic iron oxide nanoparticles (SPIONs, safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human

  1. Micro Flow Cytometry Miniaturisation - Towards in-situ Optical Phytoplankton Analysis

    Science.gov (United States)

    Zmijan, R.; Abi Kaed Bey, S.; Mowlem, M. C.; Morgan, H.

    2012-04-01

    The use of flow cytometry for studies of temporal and spatial variability of phytoplankton populations is a valuable tool contributing to research relating carbon biogeochemistry and climate change. Early designs and marine deployments of such devices started over two decades ago [1-3]. Miniaturisation and cost reduction without sacrificing performance remains a major challenge but would enable mass production and deployment. Large numbers of measurement nodes (e.g. as part of a global ocean observation system) would be possible which would increase data available over both spatial and temporal scales. This research presents two different design approaches for miniaturisation and integration of optics into a microfluidic cytometer chip. The proposed solutions are suitable for micro cytometers with external components coupled with optical fibres and were simulated and optimised using ray tracing software (Zemax). The two designs address light delivery for excitation of particles within the measurement region of the cytometer. One uses an integrated micro lens (fabricated in the chip) and the other a ball shaped micro lens manufactured separately and then inserted into the chip. Both approaches collimate the excitation light beam (from an off chip diode laser coupled with an optical fibre) into the fluidic channel. The predicted (by ray tracing) excitation beam widths are 70 and 80 µm for the integrated and the ball lens respectively, and are in agreement with experimental data presented. The proposed cytometer chip design is compatible with low cost materials (acrylic glass, cyclo-olefines) and manufacturing methods (micro milling, hot embossing, injection moulding). 1. Dubelaar, G.B.J. and P.L. Gerritzen, CytoBuoy: a step forward towards using flow cytometry in operational oceanography. Scientia Marina, 2000. 64(2): p. 255-265. 2. Peeters, J.C.H., et al., Optical Plankton Analyzer - a Flow Cytometer for Plankton Analysis .1. Design Considerations. Cytometry, 1989

  2. Cutaneous T cell lymphoma mimicking cutaneous histiocytosis: differentiation by flow cytometry.

    Science.gov (United States)

    Baines, S J; McCormick, D; McInnes, E; Dunn, J K; Dobson, J M; McConnell, I

    2000-07-01

    A two-year-old, neutered female cross-bred labrador had multiple cutaneous nodules, biopsies of which revealed pathological changes consistent with cutaneous histiocytosis. During a period of one month the dog developed multicentric lymphadenopathy, a retrobulbar mass and masses within the quadriceps and cervical muscles. Fine needle aspiration cytology of the cutaneous nodules and lymph nodes and histological examination of the cutaneous nodules and muscle masses suggested the presence of lymphoblastic lymphoma. A definitive diagnosis of CD8+ T cell lymphoma was achieved by immunophenotyping the tumour cells by flow cytometry.

  3. A structured population modeling framework for quantifying and predicting gene expression noise in flow cytometry data.

    Science.gov (United States)

    Flores, Kevin B

    2013-07-01

    We formulated a structured population model with distributed parameters to identify mechanisms that contribute to gene expression noise in time-dependent flow cytometry data. The model was validated using cell population-level gene expression data from two experiments with synthetically engineered eukaryotic cells. Our model captures the qualitative noise features of both experiments and accurately fit the data from the first experiment. Our results suggest that cellular switching between high and low expression states and transcriptional re-initiation are important factors needed to accurately describe gene expression noise with a structured population model.

  4. Diurnal Variations of Circulating Extracellular Vesicles Measured by Nano Flow Cytometry

    Science.gov (United States)

    Tigges, John; Toxavidis, Vasilis; Camacho, Virginia; Felton, Edward J.; Khoory, Joseph; Kreimer, Simion; Ivanov, Alexander R.; Mantel, Pierre-Yves; Jones, Jennifer; Akuthota, Praveen; Das, Saumya; Ghiran, Ionita

    2016-01-01

    The identification of extracellular vesicles (EVs) as intercellular conveyors of biological information has recently emerged as a novel paradigm in signaling, leading to the exploitation of EVs and their contents as biomarkers of various diseases. However, whether there are diurnal variations in the size, number, and tissue of origin of blood EVs is currently not known, and could have significant implications when using EVs as biomarkers for disease progression. Currently available technologies for the measurement of EV size and number are either time consuming, require specialized equipment, or lack sufficient accuracy across a range of EV sizes. Flow cytometry represents an attractive alternative to these methods; however, traditional flow cytometers are only capable of measuring particles down to 500 nm, which is significantly larger than the average and median sizes of plasma EVs. Utilizing a Beckman Coulter MoFlo XDP flow cytometer with NanoView module, we employed nanoscale flow cytometry (termed nanoFCM) to examine the relative number and scatter distribution of plasma EVs at three different time points during the day in 6 healthy adults. Analysis of liposomes and plasma EVs proved that nanoFCM is capable of detecting biologically-relevant vesicles down to 100 nm in size. With this high resolution configuration, we observed variations in the relative size (FSC/SSC distributions) and concentration (proportions) of EVs in healthy adult plasma across the course of a day, suggesting that there are diurnal variations in the number and size distribution of circulating EV populations. The use of nanoFCM provides a valuable tool for the study of EVs in both health and disease; however, additional refinement of nanoscale flow cytometric methods is needed for use of these instruments for quantitative particle counting and sizing. Furthermore, larger scale studies are necessary to more clearly define the diurnal variations in circulating EVs, and thus further inform

  5. Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

    Science.gov (United States)

    Verthé, Kristof; Verstraete, Willy

    2006-09-01

    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.

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

    Science.gov (United States)

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

    2016-12-01

    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.

  7. Tree-Based Methods for Discovery of Association between Flow Cytometry Data and Clinical Endpoints.

    Science.gov (United States)

    Eliot, M; Azzoni, L; Firnhaber, C; Stevens, W; Glencross, D K; Sanne, I; Montaner, L J; Foulkes, A S

    2009-01-01

    We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry: classification and regression trees (CARTs), random forests (RFs), and logic regression (LR). Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200 and 350 cell/muL. A comparison to a more standard contingency table analysis is provided. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. In all cases considered, baseline CD3-DR-CD56+CD16+ emerges as an important predictor variable, while the tree-based approaches identify additional variables as potentially informative. Application of tree-based methods to our data suggests that a combination of baseline immune activation states, with emphasis on CD8 T-cell activation, may be a better predictor than any single T-cell/innate cell subset analyzed. Taken together, we show that tree-based methods can be successfully applied to flow cytometry data to better inform and discover associations that may not emerge in the context of a univariate analysis.

  8. Tree-Based Methods for Discovery of Association between Flow Cytometry Data and Clinical Endpoints

    Directory of Open Access Journals (Sweden)

    M. Eliot

    2009-01-01

    Full Text Available We demonstrate the application and comparative interpretations of three tree-based algorithms for the analysis of data arising from flow cytometry: classification and regression trees (CARTs, random forests (RFs, and logic regression (LR. Specifically, we consider the question of what best predicts CD4 T-cell recovery in HIV-1 infected persons starting antiretroviral therapy with CD4 count between 200 and 350 cell/μL. A comparison to a more standard contingency table analysis is provided. While contingency table analysis and RFs provide information on the importance of each potential predictor variable, CART and LR offer additional insight into the combinations of variables that together are predictive of the outcome. In all cases considered, baseline CD3-DR-CD56+CD16+ emerges as an important predictor variable, while the tree-based approaches identify additional variables as potentially informative. Application of tree-based methods to our data suggests that a combination of baseline immune activation states, with emphasis on CD8 T-cell activation, may be a better predictor than any single T-cell/innate cell subset analyzed. Taken together, we show that tree-based methods can be successfully applied to flow cytometry data to better inform and discover associations that may not emerge in the context of a univariate analysis.

  9. Quantitative studies of chicken somatotrophs during growth and development by morphometry, immunocytochemistry, and flow cytometry.

    Science.gov (United States)

    Malamed, S; Deaver, D; Perez, F; Radecki, S; Gibney, J; Scanes, C G

    1997-10-01

    Changes in the male chicken somatotroph during growth and maturation have been examined by morphometric and immunocytochemical (ICC) analysis of serial sections of the anterior pituitary gland and by flow cytometry of dispersed anterior pituitary cells. ICC showed that somatotrophs are confined to the middle and caudal thirds of the anterior pituitary gland at all ages from 5 to 26 weeks. At a given age somatotrophs are of equal size at all positions along the cephalocaudal axis of the anterior pituitary gland. However, there are age-related changes: from 5 to 11 weeks rises occur in both the mean total somatotroph volume per gland (64%) and the mean number of somatotrophs (78%), while the mean volume of the single somatotroph is unchanged. From 11 to 18 weeks the mean volume of the single somatotroph decreases 41%. From 18 to 26 weeks the mean volume of the somatotroph, the mean total somatotroph volume, and the mean number per gland do not change. Flow cytometry studies suggested that somatotrophs from adults have less growth hormone (GH) than somatotrophs from young birds. The increases in total somatotroph volume and number from 5 to 11 weeks are consistent with the rise in anterior pituitary GH reported previously. Basic quantitative morphological information about age-related changes in somatotrophs is reported here. When combined with additional facts from future work, they may explain the well-documented sharp decline in circulating GH from 5 to 11 weeks. Copyright 1997 Academic Press.

  10. Immunodetection of outer membrane proteins by flow cytometry of isolated mitochondria.

    Science.gov (United States)

    Pickles, Sarah; Arbour, Nathalie; Vande Velde, Christine

    2014-09-18

    Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

  11. The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives

    Directory of Open Access Journals (Sweden)

    Debard Anne-Lise

    2005-06-01

    Full Text Available Abstract The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c. This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making.

  12. The basophil activation test by flow cytometry: recent developments in clinical studies, standardization and emerging perspectives.

    Science.gov (United States)

    Boumiza, Radhia; Debard, Anne-Lise; Monneret, Guillaume

    2005-06-30

    The diagnosis of immediate allergy is mainly based upon an evocative clinical history, positive skin tests (gold standard) and, if available, detection of specific IgE. In some complicated cases, functional in vitro tests are necessary. The general concept of those tests is to mimic in vitro the contact between allergens and circulating basophils. The first approach to basophil functional responses was the histamine release test but this has remained controversial due to insufficient sensitivity and specificity. During recent years an increasing number of studies have demonstrated that flow cytometry is a reliable tool for monitoring basophil activation upon allergen challenge by detecting surface expression of degranulation/activation markers (CD63 or CD203c). This article reviews the recent improvements to the basophil activation test made possible by flow cytometry, focusing on the use of anti-CRTH2/DP2 antibodies for basophil recognition. On the basis of a new triple staining protocol, the basophil activation test has become a standardized tool for in vitro diagnosis of immediate allergy. It is also suitable for pharmacological studies on non-purified human basophils. Multicenter studies are now required for its clinical assessment in large patient populations and to define the cut-off values for clinical decision-making.

  13. Mathematical analysis of mis-estimation of cell subsets in flow cytometry: viability staining revisited.

    Science.gov (United States)

    Petrunkina, A M; Harrison, R A P

    2011-05-31

    Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.

  14. Performance of computer vision in vivo flow cytometry with low fluorescence contrast

    Science.gov (United States)

    Markovic, Stacey; Li, Siyuan; Niedre, Mark

    2015-03-01

    Detection and enumeration of circulating cells in the bloodstream of small animals are important in many areas of preclinical biomedical research, including cancer metastasis, immunology, and reproductive medicine. Optical in vivo flow cytometry (IVFC) represents a class of technologies that allow noninvasive and continuous enumeration of circulating cells without drawing blood samples. We recently developed a technique termed computer vision in vivo flow cytometry (CV-IVFC) that uses a high-sensitivity fluorescence camera and an automated computer vision algorithm to interrogate relatively large circulating blood volumes in the ear of a mouse. We detected circulating cells at concentrations as low as 20 cells/mL. In the present work, we characterized the performance of CV-IVFC with low-contrast imaging conditions with (1) weak cell fluorescent labeling using cell-simulating fluorescent microspheres with varying brightness and (2) high background tissue autofluorescence by varying autofluorescence properties of optical phantoms. Our analysis indicates that CV-IVFC can robustly track and enumerate circulating cells with at least 50% sensitivity even in conditions with two orders of magnitude degraded contrast than our previous in vivo work. These results support the significant potential utility of CV-IVFC in a wide range of in vivo biological models.

  15. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    Science.gov (United States)

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  16. Viability and membrane potential analysis of Bacillus megaterium cells by impedance flow cytometry.

    Science.gov (United States)

    David, F; Hebeisen, M; Schade, G; Franco-Lara, E; Di Berardino, M

    2012-02-01

    Single cell analysis is an important tool to gain deeper insights into microbial physiology for the characterization and optimization of bioprocesses. In this study a novel single cell analysis technique was applied for estimating viability and membrane potential (MP) of Bacillus megaterium cells cultured in minimal medium. Its measurement principle is based on the analysis of the electrical cell properties and is called impedance flow cytometry (IFC). Comparatively, state-of-the-art fluorescence-based flow cytometry (FCM) was used to verify the results obtained by IFC. Viability and MP analyses were performed with cells at different well-defined growth stages, focusing mainly on exponential and stationary phase cells, as well as on dead cells. This was done by PI and DiOC(2)(3) staining assays in FCM and by impedance measurements at 0.5 and 10 MHz in IFC. In addition, transition growth stages of long-term cultures and agar plate colonies were characterized with both methods. FCM and IFC analyses of all experiments gave comparable results, quantitatively and qualitatively, indicating that IFC is an equivalent technique to FCM for the study of physiological cell states of bacteria. Copyright © 2011 Wiley Periodicals, Inc.

  17. Characteristics of calves produced with sperm sexed by flow cytometry/cell sorting.

    Science.gov (United States)

    Tubman, L M; Brink, Z; Suh, T K; Seidel, G E

    2004-04-01

    The objectives of this study were to determine whether calves produced by sexed sperm differed from controls and to what extent the sex ratio of calves was altered by the sexing procedure. Data were collected from 1,169 calves produced from sperm sexed by flow cytometry/cell sorting after staining with Hoechst 33342, and 793 calves produced from control sperm during breeding trials between 1997 and 2001. Least squares ANOVA were completed using factors of treatment (sexed vs. control sperm), 19 management groups from 13 field trials, and calf sex. Responses analyzed include gestation length, birth weight, calving ease, calf vigor, weaning weight, abortion rate, and death rates (neonatal and through weaning). No significant difference was observed for any response due to treatment or treatment interactions (P > 0.10). Therefore, calves produced from sexed sperm grew and developed normally both pre- and postnatally. A neurological disorder was observed in four control calves and one sexed calf from one farm. No gross anatomical abnormalities were reported for any calves in the study. Differences were observed for all responses among management groups (P Flow cytometry/cell sorting can be used to preselect sex of calves safely with approximately 90% accuracy.

  18. Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities.

    Science.gov (United States)

    Müller, Susann; Nebe-von-Caron, Gerhard

    2010-07-01

    The still poorly explored world of microbial functioning is about to be uncovered by a combined application of old and new technologies. Bacteria, especially, are still in the dark with respect to their phylogenetic affiliations as well as their metabolic capabilities and functions. However, with the advent of sophisticated flow cytometric and cell sorting technologies in microbiological labs, there is now the possibility to gain this knowledge at the single-cell level without cumbersome cultivation approaches. Cytometry also facilitates the understanding of physiological diversity in seemingly likewise acting populations. Both individuality and diversity lead to the complex and concerted actions of microbial consortia. This review provides an overview of the state of the art in the field. It deals with the handling of microorganisms from the very beginning (i.e. sampling, and detachment and fixation procedures) and goes on to discuss the pitfalls and problems in analysing cells without any further treatment. If information cannot be gained by specific staining procedures, phylogenetic technologies, transcriptomic and proteomic approaches may be options for achieving advanced insights. All in all, flow cytometry will be a mediator technology to gain a deeper insight into the heterogeneity of populations and the functioning of microbial communities.

  19. Recent advances on multi-parameter flow cytometry to characterize antimicrobial treatments

    Directory of Open Access Journals (Sweden)

    Lucie LEONARD

    2016-08-01

    Full Text Available The investigation on antimicrobial mechanisms is a challenging and crucial issue in the fields of food or clinical microbiology, as it constitutes a prerequisite to the development of new antimicrobial processes or compounds, as well as to anticipate phenomenon of microbial resistance. Nowadays it is accepted that a cells population exposed to a stress can cause the appearance of different cell populations and in particular sub-lethally compromised cells which could be defined as viable but non culturable (VBNC. Recent advances on flow cytometry (FCM and especially on multi-parameter flow cytometry (MP-FCM provide the opportunity to obtain high-speed information at real time on damage at single-cell level. This review gathers MP-FCM methodologies based on individual and simultaneous staining of microbial cells employed to investigate their physiological state following different physical and chemical antimicrobial treatments. Special attention will be paid to recent studies exploiting the possibility to corroborate MP-FCM results with additional techniques (plate counting, microscopy, spectroscopy, molecular biology techniques, membrane modeling in order to elucidate the antimicrobial mechanism of action of a given antimicrobial treatment or compound. The combination of MP-FCM methodologies with these additional methods is namely a promising and increasingly used approach to give further insight in differences in microbial sub-population evolutions in response to antimicrobial treatments.

  20. Discriminative variable subsets in Bayesian classification with mixture models, with application in flow cytometry studies.

    Science.gov (United States)

    Lin, Lin; Chan, Cliburn; West, Mike

    2016-01-01

    We discuss the evaluation of subsets of variables for the discriminative evidence they provide in multivariate mixture modeling for classification. The novel development of Bayesian classification analysis presented is partly motivated by problems of design and selection of variables in biomolecular studies, particularly involving widely used assays of large-scale single-cell data generated using flow cytometry technology. For such studies and for mixture modeling generally, we define discriminative analysis that overlays fitted mixture models using a natural measure of concordance between mixture component densities, and define an effective and computationally feasible method for assessing and prioritizing subsets of variables according to their roles in discrimination of one or more mixture components. We relate the new discriminative information measures to Bayesian classification probabilities and error rates, and exemplify their use in Bayesian analysis of Dirichlet process mixture models fitted via Markov chain Monte Carlo methods as well as using a novel Bayesian expectation-maximization algorithm. We present a series of theoretical and simulated data examples to fix concepts and exhibit the utility of the approach, and compare with prior approaches. We demonstrate application in the context of automatic classification and discriminative variable selection in high-throughput systems biology using large flow cytometry datasets.

  1. Evaluating the morphology of erythrocyte population: An approach based on atomic force microscopy and flow cytometry.

    Science.gov (United States)

    Ghosh, Sayari; Chakraborty, Ishita; Chakraborty, Monojit; Mukhopadhyay, Ashis; Mishra, Raghwendra; Sarkar, Debasish

    2016-04-01

    Erythrocyte morphology is gaining importance as a powerful pathological index in identifying the severity of any blood related disease. However, the existing technique of quantitative microscopy is highly time consuming and prone to personalized bias. On the other hand, relatively unexplored, complementary technique based on flow cytometry has not been standardized till date, particularly due to the lack of a proper morphological scoring scale. In this article, we have presented a new approach to formulate a non-empirical scoring scale based on membrane roughness (R(rms)) data obtained from atomic force microscopy. Subsequently, the respective morphological quantifier of the whole erythrocyte population, commonly known as morphological index, was expressed as a function of highest correlated statistical parameters of scattered signal profiles generated by flow cytometry. Feed forward artificial neural network model with multilayer perceptron architecture was used to develop the intended functional form. High correlation coefficient (R(2) = 0.95), even for model-formulation exclusive samples, clearly indicates the universal validity of the proposed model. Moreover, a direct pathological application of the proposed model has been illustrated in relation to patients, diagnosed to be suffering from a wide variety of cancer.

  2. Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry.

    Science.gov (United States)

    Loureiro, J; Pinto, G; Lopes, T; Dolezel, J; Santos, C

    2005-08-01

    Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.

  3. Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

    Science.gov (United States)

    García-Varela, Rebeca; García-García, Rebeca M; Barba-Dávila, Bertha A; Fajardo-Ramírez, Oscar R; Serna-Saldívar, Sergio O; Cardineau, Guy A

    2015-10-14

    Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

  4. Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Rebeca García-Varela

    2015-10-01

    Full Text Available Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar, in a range from 1 to 4 μg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

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

    Science.gov (United States)

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

    2013-03-01

    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.

  6. Enhanced red and near infrared detection in flow cytometry using avalanche photodiodes.

    Science.gov (United States)

    Lawrence, William G; Varadi, Gyula; Entine, Gerald; Podniesinski, Edward; Wallace, Paul K

    2008-08-01

    Polychromatic flow cytometry enables detailed identification of cell phenotype using multiple fluorescent parameters. The photomultiplier tubes (PMTs) used to detect fluorescence in current instruments limit the sensitivity in the long wavelength spectral range. We demonstrate the flow cytometric applications of silicon avalanche photodiodes (APDs), which have improved red sensitivity and a working fluorescence detection range beyond 1,000 nm. A comparison of the wavelength-dependent performance of the APD and PMT was carried out using pulsed light-emitting diode sources, calibrated test beads, and biological samples. A breadboard flow cytometer test bench was constructed to compare the performance of PMTs and APD detectors. The APD used an additional amplifier stage to match the internal gain of the PMT. The resolution of the APD and PMT was compared for flow cytometry applications using a pulsed light-emitting diode source over the 500-1060 nm spectral range. These measurements showed the relative changes in the signal-to-noise performance of the APD and PMT over a broad spectral range. Both the APD and PMTs were used to measure the signal-to-noise response for a set of six peak calibration beads over the 530-800 nm wavelength range. CD4-positive cells labeled with antibody-conjugated phycoerythrin or 800 nm quantum dots were identified by simultaneous detection using the APD and the PMT. The ratios of the intensities of the CD4- and CD4+ populations were found to be similar for both detectors in the visible wavelengths, but only the APD was able to separate these populations at wavelengths above 800 nm. These measurements illustrate the differences in APD and PMT performance at different wavelengths and signal intensity levels. While the APD and PMT show similar signal-to-noise performance in the visible spectral range, the dark noise of the APD detector reduces the sensitivity at low signal levels. At wavelengths longer than 650 nm, the high quantum efficiency

  7. Minimal residual disease monitoring by 8-color flow cytometry in mantle cell lymphoma: an EU-MCL and LYSA study

    Science.gov (United States)

    Cheminant, Morgane; Derrieux, Coralie; Touzart, Aurore; Schmit, Stéphanie; Grenier, Adrien; Trinquand, Amélie; Delfau-Larue, Marie-Hélène; Lhermitte, Ludovic; Thieblemont, Catherine; Ribrag, Vincent; Cheze, Stéphane; Sanhes, Laurence; Jardin, Fabrice; Lefrère, François; Delarue, Richard; Hoster, Eva; Dreyling, Martin; Asnafi, Vahid; Hermine, Olivier; Macintyre, Elizabeth

    2016-01-01

    Quantification of minimal residual disease may guide therapeutic strategies in mantle cell lymphoma. While multiparameter flow cytometry is used for diagnosis, the gold standard method for minimal residual disease analysis is real-time quantitative polymerase chain reaction (RQ-PCR). In this European Mantle Cell Lymphoma network (EU-MCL) pilot study, we compared flow cytometry with RQ-PCR for minimal residual disease detection. Of 113 patients with at least one minimal residual disease sample, RQ-PCR was applicable in 97 (86%). A total of 284 minimal residual disease samples from 61 patients were analyzed in parallel by flow cytometry and RQ-PCR. A single, 8-color, 10-antibody flow cytometry tube allowed specific minimal residual disease assessment in all patients, with a robust sensitivity of 0.01%. Using this cut-off level, the true-positive-rate of flow cytometry with respect to RQ-PCR was 80%, whereas the true-negative-rate was 92%. As expected, RQ-PCR frequently detected positivity below this 0.01% threshold, which is insufficiently sensitive for prognostic evaluation and would ideally be replaced with robust quantification down to a 0.001% (10-5) threshold. In 10 relapsing patients, the transition from negative to positive by RQ-PCR (median 22.5 months before relapse) nearly always preceded transition by flow cytometry (4.5 months), but transition to RQ-PCR positivity above 0.01% (5 months) was simultaneous. Pre-emptive rituximab treatment of 2 patients at minimal residual disease relapse allowed re-establishment of molecular and phenotypic complete remission. Flow cytometry minimal residual disease is a complementary approach to RQ-PCR and a promising tool in individual mantle cell lymphoma therapeutic management. PMID:26703963

  8. Improved and Reproducible Flow Cytometry Methodology for Nuclei Isolation from Single Root Meristem

    Directory of Open Access Journals (Sweden)

    Thaís Cristina Ribeiro Silva

    2010-01-01

    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.

  9. Basophil activation test by flow cytometry: present and future applications in allergology.

    Science.gov (United States)

    Ebo, D G; Bridts, C H; Hagendorens, M M; Aerts, N E; De Clerck, L S; Stevens, W J

    2008-07-01

    The diagnosis of allergic reactions in clinical practice rests upon both clinical history and the demonstration of specific immunoglobulin E (sIgE), either in the serum or via skin tests. However, for various reasons, identification of the offending allergen(s) is not always possible. Moreover, not all allergies are IgE-mediated. In an attempt to find reliable methods to investigate hypersensitivity reactions, histamine and sulfidoleukotriene release tests have long been introduced. However, relatively few comprehensive quality reports have been published so far. Upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. This review addresses the principals, particular technical aspects and pitfalls as well as the clinical and research applications of flow-assisted analysis of in vitro activated basophils.

  10. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.

    Science.gov (United States)

    Malleret, Benoît; Claser, Carla; Ong, Alice Soh Meoy; Suwanarusk, Rossarin; Sriprawat, Kanlaya; Howland, Shanshan Wu; Russell, Bruce; Nosten, Francois; Rénia, Laurent

    2011-01-01

    Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing.

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

    Science.gov (United States)

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

    2002-08-15

    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.

  12. Use of flow cytometry for high-throughput cell population estimates in fixed brain tissue

    Directory of Open Access Journals (Sweden)

    Nicole A Young

    2012-07-01

    Full Text Available The numbers and types of cells in an area of cortex define its function. Therefore it is essential to characterize the numbers and distributions of total cells in areas of the cortex, as well as to identify numbers of subclasses of neurons and glial cells. To date, the large size of the primate brain and the lack of innovation in cell counting methods have been a roadblock to obtaining high-resolution maps of cell and neuron density across the cortex in humans and non-human primates. Stereological counting methods and the isotropic fractionator are valuable tools for estimating cell numbers, but are better suited to smaller, well-defined brain structures or to cortex as a whole. In the present study, we have extended our flow-cytometry based counting method, the flow fractionator (Collins et al., 2010a, to include high-throughput total cell population estimates in homogenized cortical samples. We demonstrate that our method produces consistent, accurate and repeatable cell estimates quickly. The estimates we report are in excellent agreement with estimates for the same samples obtained using a Neubauer chamber and a fluorescence microscope. We show that our flow cytometry-based method for total cell estimation in homogenized brain tissue is more efficient and more precise than manual counting methods. The addition of automated nuclei counting to our flow fractionator method allows for a fully automated, rapid characterization of total cells and neuronal and non-neuronal populations in human and non-human primate brains, providing valuable data to further our understanding of the functional organization of normal, aging and diseased brains.

  13. A novel fluorescent sensor for measurement of CFTR function by flow cytometry.

    Science.gov (United States)

    Vijftigschild, Lodewijk A W; van der Ent, Cornelis K; Beekman, Jeffrey M

    2013-06-01

    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.

  14. Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes.

    Science.gov (United States)

    Webb, Craig; Bedwell, Cathy; Guth, Amanda; Avery, Paul; Dow, Steven

    2006-08-15

    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.

  15. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    Science.gov (United States)

    Bono, Michael S; Garcia, Ravi D; Sri-Jayantha, Dylan V; Ahner, Beth A; Kirby, Brian J

    2015-01-01

    In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  16. Measurement of lipid accumulation in Chlorella vulgaris via flow cytometry and liquid-state ¹H NMR spectroscopy for development of an NMR-traceable flow cytometry protocol.

    Directory of Open Access Journals (Sweden)

    Michael S Bono

    Full Text Available In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.

  17. Quantification of intracellular polyhydroxyalkanoates by virtue of personalized flow cytometry protocol.

    Science.gov (United States)

    Saranya, V; Poornimakkani; Krishnakumari, M S; Suguna, P; Binuramesh, C; Abirami, P; Rajeswari, V; Ramachandran, K B; Shenbagarathai, R

    2012-11-01

    Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microbes, a potential alternative to synthetic plastics. Various methods ranging from gravimetry to spectrophotometry are routinely used for qualitative analysis of extracted PHA. There is a great need for accurate quantification of intracellular PHA during bioprocess. Hence, the present study aims to improvise the existing Nile red-based flow cytometry protocol. It was achieved using respective cells in a non-PHA accumulating state as gating control to minimize non-specific staining. The optimal Nile red concentration required for PHA staining is 5 × 10(3) pg mL(-1), which is ~10(3)-fold less than that of earlier reports. Further, it was inferred that flow-based quantification was more accurate than the gravimetric method. The intracellular PHA content was highest in Pseudomonas sp. MNNG-S (52.06 %) among the Pseudomonas strains tested by the flow-based method. Both gravimetric and flow-based cell cycle analyses revealed that DNA synthesis (S phase) and PHA production (log phase) are synchronous at 24-48 h of culture. This study supports flow-based PHA quantification for real time online measurement of intracellular PHA for bioreactor monitoring, control and optimization enduing industrial applications.

  18. Study of inertial hydrodynamic focusing in sheath-driven flows for lab-on-a-chip flow cytometry

    Science.gov (United States)

    Panwar, Nishtha; Song, Peiyi; Yong, Ken-Tye; Tjin, Swee Chuan

    2017-05-01

    Miniature flow cytometer models enable fast and cost-effective management of diseases in vulnerable and low-end settings. The single-line focusing of cell or particle samples is achieved using hydrodynamic forces in the microfluidic channels. The two common configurations among them are the single-sheath and dual-sheath flows wherein the sample is directed through the main channel, and the surrounding sheath fluids are directed into the main channel through inlets on either side of the main channel. Most models predict the width of the focused sample stream based on hydrodynamic focusing in the low Reynolds number regime (Re << 1), where the viscous forces dominate the inertial forces. In this work, we present comparative analysis of particle focusing by single-sheath and dual-sheath configurations for focusing of micron-sized cells/particles in the range 2 to 20 μm in the higher Re (10 < Re < 80) laminar regime. A quantitative analysis of the relative focused stream width (wf/wch) as a function of flow rate ratio (FRR = Sample flow rate/Sheath flow rate) for the two configurations is presented. The particle tracing results are also compared with the experimental fluorescent microscopy results at various FRR. The deviations of the results from the theoretical predictions of hydrodynamic focusing at Re << 1, are explained analytically. These findings clearly outline the range of flow parameters and relative particle sizes that can be used for cytometry studies for a given channel geometry. This is a highly predictive modeling method as it provides substantial results of particle positions across the microchannel width according to their size and FRR for single-line focusing of particles. Such information is crucial for one to engineer miniaturized flow cytometry for screening of desired cells or particles.

  19. Characterization of viability and proliferation of alginate-poly-L-lysine-alginate encapsulated myoblasts using flow cytometry.

    Science.gov (United States)

    Thakur, Ajit; Sengupta, Ruchira; Matsui, Hideto; Lillicrap, David; Jones, Kim; Hortelano, Gonzalo

    2010-08-01

    Genetically modified cells encapsulated in alginate-poly-L-lysine-alginate (APA) are being developed to deliver therapeutic products to treat a variety of diseases. The characterization of the encapsulated cells thus becomes paramount. This study reports a novel method to assess the viability, granularity and proliferation of encapsulated cells based on flow cytometry. The in vitro viability of encapsulated G8 murine myoblasts secreting canine FVIII (cFVIII) measured by flow cytometry was comparable to the traditional trypan blue exclusion method and both correlated with cFVIII secretion levels. In contrast, after implantation into mice, only viability measured by flow cytometry correlated with cFVIII secretion. Further, flow cytometry analysis of encapsulated cells maintained in vitro and in vivo revealed a greater fraction of granular cells compared to free cells, suggesting that encapsulation influences the morphology (cytoplasmic composition) of cells within APA microcapsules. Interestingly, the proliferation study showed that encapsulated cells proliferate faster, on average, and were more heterogeneous in vivo compared to in vitro culture conditions, suggesting that encapsulated cell proliferation is complex and environment-dependent. In conclusion, we show that flow cytometry analysis allows for a more consistent and comprehensive examination of encapsulated cells to aid in the development of cell therapy protocols.

  20. An open-source solution for advanced imaging flow cytometry data analysis using machine learning.

    Science.gov (United States)

    Hennig, Holger; Rees, Paul; Blasi, Thomas; Kamentsky, Lee; Hung, Jane; Dao, David; Carpenter, Anne E; Filby, Andrew

    2017-01-01

    Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using "user-friendly" platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data sets. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery.

  1. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

    Energy Technology Data Exchange (ETDEWEB)

    Pellicciari, C.; Mangiarotti, R.; Bottone, M.G.; Danova, M. [Univ. of Pavia (Italy); Wang, E. [Jewish General Hospital, Montreal, Quebec (Canada)

    1995-12-01

    Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.

  2. Optical analysis of nanomaterial-cell interactions: flow cytometry and digital holographic microscopy

    Science.gov (United States)

    Mues, Sarah; Antunovic, Jan; Ossig, Rainer; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    The in vitro cytotoxicity assessment of engineered nanoparticles commonly involves the measurement of different endpoints like the formation of reactive oxygen species, cell viability or cell death. Usually these parameters are determined by optical readouts of enzymatically converted substrates that often interfere with the tested nanomaterials. Using cell viability (WST-8) and cell death (LDH) as parameter we have initially investigated the toxic effects of spherical (NM 300) and rod shaped (NM 302) silver nanomaterials with a matrix of four cell lines representing different functions: lung and kidney epithelial cells, macrophages and fibroblasts. In addition, we have used a label-free flow cytometer configuration to investigate interactions of particles and macrophages by side scatter signal analysis. Finally, we explored digital holographic microscopy (DHM) for multimodal label-free analysis of nanomaterial toxicity. Quantitative DHM phase images were analyzed for cell thickness, volume, density, dry mass and refractive index. We could demonstrate that silver spheres lead to more cytotoxic effects than rods in all four examined cell lines and both assay. Exemplarily a dose dependent interaction increase of cells with NM 300 and NM 302 analyzed by flow cytometry is shown. Furthermore, we found that the refractive index of cells is influenced by incubation with NM 300 in a decreasing manner. A 24 hours time-lapse measurement revealed a dose dependent decrease of dry mass and surface area development indicating reduced cell viability and cell death. Our results demonstrate digital holographic microscopy and flow cytometry as valuable label-free tools for nanomaterial toxicity and cell interaction studies.

  3. Improved graft survival in highly sensitized patients undergoing renal transplantation after the introduction of a clinically validated flow cytometry crossmatch.

    LENUS (Irish Health Repository)

    Limaye, Sandhya

    2009-04-15

    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.

  4. Flow cytometry quality requirements for monitoring of minimal disease in plasma cell myeloma.

    Science.gov (United States)

    Oldaker, Teri A; Wallace, Paul K; Barnett, David

    2016-01-01

    Current therapeutic approaches for plasma cell myeloma (PCM) attain an overall survival of more than 6 years for the majority of newly diagnosed patients. However, PFS and OS are the only accepted FDA clinical endpoints for demonstrating drug efficacy before they can be become frontline therapeutic options. There is, however, recognition that the increasing gap between drug development and approval for mainstream therapeutic use needs to be shortened. As such regulatory bodies such as the FDA are now considering whether biomarker response evaluation, as in measurement of minimal residual disease (MRD) as assessed by flow cytometry (FC), can provide an early, robust prediction of survival and therefore improve the drug approval process. Recently, FC MRD using a standardized eight-color antibody methodology has been shown to have a minimum sensitivity of 0.01% and an upper sensitivity of 0.001%. To ensure that all laboratories using this approach achieve the same levels of sensitivity it is crucially important to have standardized quality management procedures in place. This manuscript accompanies those published in this special issue and describes the minimum that is required for validating and quality monitoring of this highly specific test to ensure any laboratory, irrespective of location, will achieve the expected quality standards required. © 2015 International Clinical Cytometry Society.

  5. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    Science.gov (United States)

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

  6. Impedance flow cytometry gauges proliferative capacity by detecting TRPC1 expression.

    Science.gov (United States)

    Crocetti, Sara; Beyer, Christian; Unternährer, Silvio; Benavides Damm, Tatiana; Schade-Kampmann, Grit; Hebeisen, Monika; Di Berardino, Marco; Fröhlich, Jürg; Franco-Obregón, Alfredo

    2014-06-01

    When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedance-based flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation. © 2014 International Society for Advancement of Cytometry.

  7. Impact of reticulated platelets on antiplatelet response to thienopyridines is independent of platelet turnover.

    Science.gov (United States)

    Stratz, Christian; Nührenberg, Thomas; Amann, Michael; Cederqvist, Marco; Kleiner, Pascal; Valina, Christian M; Trenk, Dietmar; Neumann, Franz-Josef; Hochholzer, Willibald

    2016-10-28

    Reticulated platelets are associated with impaired antiplatelet response to thienopyridines. It is uncertain whether this interaction is caused by a decreased drug exposure due to high platelet turnover reflected by elevated levels of reticulated platelets or by intrinsic properties of reticulated platelets. This study sought to investigate if the impact of reticulated platelets on early antiplatelet response to thienopyridines is mainly caused by platelet turnover as previously suggested. Elective patients undergoing coronary intervention were randomised to loading with clopidogrel 600 mg or prasugrel 60 mg (n=200). Adenosine diphosphate (ADP)-induced platelet reactivity was determined by impedance aggregometry before, at 30, 60, 90, and 120 minutes and at day 1 after loading. Immature platelet count was assessed as marker of reticulated platelets by flow cytometry. Platelet reactivity increased with rising levels of immature platelet count in both groups. This effect was more distinctive in patients on clopidogrel as compared to patients on prasugrel. Overall, immature platelet count correlated well with on-treatment platelet reactivity at all time-points (p < 0.001). These correlations did not change over time in the entire cohort as well as in patients treated with clopidogrel or prasugrel indicating an effect independent of platelet turnover (comparison of correlations 120 minutes/day 1: p = 0.64). In conclusion, the association of immature platelet count with impaired antiplatelet response to thienopyridines is similar early and late after loading. This finding suggests as main underlying mechanism another effect of reticulated platelets on thienopyridines than platelet turnover.

  8. Light-scattering flow cytometry for identification and characterization of blood microparticles.

    Science.gov (United States)

    Konokhova, Anastasiya I; Yurkin, Maxim A; Moskalensky, Alexander E; Chernyshev, Andrei V; Tsvetovskaya, Galina A; Chikova, Elena D; Maltsev, Valeri P

    2012-05-01

    We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 μm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 μm due to used laser wavelength of 0.66 μm.

  9. Light-scattering flow cytometry for identification and characterization of blood microparticles

    Science.gov (United States)

    Konokhova, Anastasiya I.; Yurkin, Maxim A.; Moskalensky, Alexander E.; Chernyshev, Andrei V.; Tsvetovskaya, Galina A.; Chikova, Elena D.; Maltsev, Valeri P.

    2012-05-01

    We describe a novel approach to study blood microparticles using the scanning flow cytometer, which measures light scattering patterns (LSPs) of individual particles. Starting from platelet-rich plasma, we separated spherical microparticles from non-spherical plasma constituents, such as platelets and cell debris, based on similarity of their LSP to that of sphere. This provides a label-free method for identification (detection) of microparticles, including those larger than 1 μm. Next, we rigorously characterized each measured particle, determining its size and refractive index including errors of these estimates. Finally, we employed a deconvolution algorithm to determine size and refractive index distributions of the whole population of microparticles, accounting for largely different reliability of individual measurements. Developed methods were tested on a blood sample of a healthy donor, resulting in good agreement with literature data. The only limitation of this approach is size detection limit, which is currently about 0.5 μm due to used laser wavelength of 0.66 μm.

  10. Comparison of urine cell characteristics by flow cytometry and cytology in patients suspected of having bladder cancer.

    Science.gov (United States)

    Barlandas-Rendón, Eric; Müller, Mathias M; García-Latorre, Ethel; Heinschink, Angelika

    2002-08-01

    The diagnosis of bladder cancer is confirmed by histological analysis of tissue biopsies. Cytology of urine samples is a noninvasive alternative. The aim of this work was to find out whether flow cytometry of urine samples is more sensitive than cytology. For this purpose we studied 115 patients suspected of having bladder cancer. Cells isolated from urine samples were analyzed by cytometry for the expression of cytokeratin and CD 45 and for DNA measurements such as: DNA index, synthesis phase fraction and proliferative index (SPF + G2/M phase). At the same time we carried out cytological analysis. All positive cases were confirmed by histology (21/115), 18 were diagnosed by flow cytometry and 16 by cytology, with a sensitivity of 85.7% and 76.1%, respectively. Two cases were found to be positive by flow cytometry, which were not confirmed by histology, while no false positives were detected by cytology. We found that both techniques gave almost identical results for the diagnosis of bladder cancer, although there were differences in non-malignant samples. In conclusion, flow cytometry is slightly more sensitive than cytology but the combination of the two techniques improves the diagnosis.

  11. Nuclear DNA content of the pigeon orchid (Dendrobium crumenatum Sw. with the analysis of flow cytometry

    Directory of Open Access Journals (Sweden)

    Upatham Meesawat

    2008-05-01

    Full Text Available Nuclear DNA content for the adult plants grown in a greenhouse and in vitro young plantlets of the pigeon orchid (Dendrobium crumenatum Sw. was analyzed using flow cytometry. The resulting 2C DNA values ranged from 2.30±0.14 pgto 2.43±0.06 pg. However, nuclear DNA ploidy levels of long-term in vitro plantlets were found to be triploid and tetraploid.These ploidy levels were confirmed by chromosome counting. Tetraploid individuals (2n = 4x = 76 had approximately two times DNA content than diploid (2n = 2x = 38 individuals. This variation may be due to prolonged cultivation and thepresence of exogenous plant growth regulators.

  12. Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

    Science.gov (United States)

    Barbosa, Joana; Rodrigues, Acácio Gonçalves; Pérez, Maria José; Pina-Vaz, Cidália

    2014-01-01

    Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability. PMID:25424449

  13. A Triple Staining Method for Accurate Cell Cycle Analysis Using Multiparameter Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Lin Qiu

    2013-12-01

    Full Text Available Cell cycle analysis is important for cancer research. We present herein a novel method for accurate cell cycle analysis. This method analyzes the cell cycle by multiparameter flow cytometry based on simultaneously labeling the cell nuclear DNA, RNA, and phosphorylated mitotic nuclei protein, using Hoechst 33342, pyronin Y, and MPM-2-Cy5, respectively, and our results demonstrated that this method could effectively divide the cell cycle into G0, G1, S, G2, and M phases. We further tested this method using the clinical anticancer agents crizotinib and taxol, and the results clearly illustrated that crizotinib and taxol arrested Jurkat cells in G0 and M phase, respectively. These results indicate that this method could be a very useful tool for cytokinetic and pharmacological research.

  14. Development by flow cytometry of bioassays based on chlorella for environmental monitoring

    Directory of Open Access Journals (Sweden)

    Petrescu C-M,

    2016-05-01

    Full Text Available In ecotoxicological assessments, bioassays (ecotoxicity tests or biotests are one of the main tools, defined as methods which use living cells, tissues, organism or communities to assess exposure-related effects of chemicals. The increasing complexity of environmental degradation requires an increase in the capacity of scientific approach in monitoring and notification as early as possible risks. Our own objective concerns the detection of aquatic environment pollution in Romania and particularly in the Danube basin. For assessing aquatic environment pollution degree or for assessing cytotoxicity or ecotoxicity of pollutants (heavy metals, nanoparticles, pesticides, etc. we developed news experimental bioassays based on the use of viability and apoptosis biomarkers of Chlorella cells by flow cytometry. Our proposed bioassays could be rapid and very sensitive tests for in laboratory aquatic risk assessment and biomonitoring.

  15. Analysis of DNA-guided self-assembly of microspheres using imaging flow cytometry.

    Science.gov (United States)

    Tang, Hao; Deschner, Ryan; Allen, Peter; Cho, Younjin; Sermas, Patrick; Maurer, Alejandro; Ellington, Andrew D; Willson, C Grant

    2012-09-19

    Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium.

  16. Surface profiling of normally responding and nonreleasing basophils by flow cytometry

    DEFF Research Database (Denmark)

    Kistrup, Kasper; Poulsen, Lars Kærgaard; Jensen, Bettina Margrethe

    Background Human basophils are granulocytes with the capacity to play important roles in allergy for example by releasing histamine when activated by cross-linking of their high affinity IgE receptors (Fc¿RI). However, not all individuals have basophils responding with a histamine release after......c, C3aR, C5aR CCR3, FPR1, ST2, CRTH2 on anti-IgE respondsive and nonreleasing basophils by flow cytometry, thereby generating a surface profile of the two phenotypes. Methods Fresh buffy coat blood (histamine release and nonreleases were defined as having...... a maximum release basophils, defined as FceRIa+CD3-CD14-CD19-CD56-,were analysed for surface expression of relevant markers. All samples were compensated and analysed in logicle display. All gates...

  17. Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear]. E-mail; neylisantos@yahoo.com.br; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria [Fundacao de Hematologia e Hemoterapia de Pernambuco, Recife, PE (Brazil). Unidade de Laboratorios Especializados. Lab. de Imunofenotipagem

    2008-12-15

    The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo- and radiotherapy, particularly when radiation exposure precedes chemical treatment. (author)

  18. Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes.

    Science.gov (United States)

    Tracy, Bryan P; Gaida, Stefan M; Papoutsakis, Eleftherios T

    2010-02-01

    Flow cytometry (FC) and FC-based cell sorting have been established as critical tools in modern cell and developmental biology. Yet, their applications in bacteria, especially in the multiparametric mode, remain limited. We argue that FC technologies have the potential to greatly accelerate the analysis and development of microbial complex phenotypes through applications of metabolic engineering, synthetic biology, and evolutionary engineering. We demonstrate the importance of FC for elucidating population heterogeneity because of developmental processes or epigenetic regulation. FC can be engaged for both synthetic and analytical applications of complex phenotypes within a single species, multispecies, and microbial-library populations. Examples include methods to identify developmental microbial stages associated with productive metabolic phenotypes, select desirable promoters from a single species or metagenomic libraries, and to screen designer riboswitches for synthetic-biology applications.

  19. Evaluation of Giardia duodenalis viability after metronidazole treatment by flow cytometry

    Directory of Open Access Journals (Sweden)

    Joana Barbosa

    2014-12-01

    Full Text Available Giardia duodenalis (syn. lamblia; syn. intestinalis susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ. Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

  20. The importance of immunophenotyping by flow cytometry in distinction between hematogones and B lymphoblasts

    Directory of Open Access Journals (Sweden)

    Aline B. Wohlfahrt

    2015-02-01

    Full Text Available Hematogones are normal B-lineage lymphoid precursors in the bone marrow. B lymphoblasts are immature neoplastic cells present in patients with precursor B-cell acute lymphoblastic leukemia (B-ALL. Hematogones and B lymphoblasts share characteristics, such as morphological similarity often indistinct and expression of the same antigens in immunophenotypic analysis. Increased numbers of hematogones in patients with B-ALL during regeneration of bone marrow after treatment for leukemia, in cases of disease relapse or marrow transplantation, may be subject to questions about the nature and prognosis of this immature cell. This article presents information about the morphological and immunophenotypic characteristics of B lymphoid precursors and verifies the relevance of immunophenotyping by flow cytometry (FC in the distinction between those cells. This differentiation is essential to establish a correct prognosis and assist in medical decision about the most appropriate therapeutic scheme.

  1. Expression of Bruton's tyrosine kinase in B-cell neoplasms evaluated by flow cytometry.

    Science.gov (United States)

    Marcondes, Natália Aydos; Fernandes, Flavo Beno; Alegretti, Ana Paula; Faulhaber, Gustavo Adolpho Moreira

    2016-12-27

    Bruton's tyrosine kinase (BTK) is a cytoplasmatic protein that is part of the B-cell antigen receptor signaling pathway. Our aim was to evaluate the expression of BTK in B-cell neoplasms and compare it to normal B-cell lymphocytes. After surface staining with CD19 and CD45, flow cytometry staining for intracellular BTK was performed in leukemic or mature B-cells from bone marrow or peripheral blood samples. No differences in BTK expression were identified between groups, or in comparison to control samples, there was no association between BTK expression and the clinical variables evaluated. BTK expression in B-cell neoplasms was similar to that of normal B-cell lymphocytes.

  2. Isolation of carotenoid hyperproducing mutants of Xanthophyllomyces dendrorhous (Phaffia rhodozyma) by flow cytometry and cell sorting.

    Science.gov (United States)

    Brehm-Stecher, Byron F; Johnson, Eric A

    2012-01-01

    Approaches for improving astaxanthin yields in Xanthophyllomyces dendrorhous include optimization of fermentation conditions and generation of hyperproducing mutants through random mutagenesis using chemical or physical means. A key limitation of classical mutagenesis is the labor-intensive nature of the screening processes required to find relatively rare mutants having increased carotenoid content, as these are present against a high background of low-interest cells. Here, flow cytometry is described as a high-throughput, single-cell method for primary enrichment of mutagenized cells expressing high levels of astaxanthin. This approach improves the speed and productivity of classical strain selection, enhancing the chances for isolating the carotenoid hyperproducing mutants (CHMs) needed to enable high-titer, economical production of natural astaxanthin.

  3. CASP3 protein expression by flow cytometry in Down's syndrome subjects.

    Science.gov (United States)

    Salemi, Michele; Condorelli, Rosita A; Romano, Corrado; Concetta, Barone; Romano, Carmelo; Salluzzo, Maria Grazia; Bosco, Paolo; Calogero, Aldo E

    2014-01-01

    Down's syndrome (DS), the most common chromosomal disorder, is caused by 21 trisomy and is featured by intellectual disability. Subjects with DS can develop some traits of Alzheimer disease (AD) at an earlier age than subjects without trisomy 21. Apoptosis is a programmed cell death process under both normal physiological and pathological conditions. Caspase-3 (CASP3) plays an important role in neuronal death during nervous system development and under certain pathological conditions. Furthermore, in vitro and in vivo studies report elevated expression and activation of CASP3 in models of AD. On this account, the expression of CASP3 gene was evaluated in cultures of fibroblasts of DS and normal subjects by flow cytometry. CASP3 protein was up-regulated in fibroblasts of DS. The data obtained from this study strengthen the hypothesis that the over-expression of CASP3 gene could have a role in the activation of the apoptotic pathways acting in the neurodegenerative processes in DS.

  4. NetFCM: A Semi-Automated Web-Based Method for Flow Cytometry Data Analysis

    DEFF Research Database (Denmark)

    Frederiksen, Juliet Wairimu; Buggert, Marcus; Karlsson, Annika C.

    2014-01-01

    data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online...... corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. (c) 2014 International Society......Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM...

  5. Development of an Immunomagnetic Separation Method for Viable Salmonella Typhimurium Detected by Flow Cytometry

    DEFF Research Database (Denmark)

    Ahmed, Shakil; Rubahn, Horst-Günter; Erdmann, Helmut

    2016-01-01

    for detection of food-related bacteria. In this study, a flow cytometry based immunomagnetic separation (IMS) method for the isolation and enrichment of Salmonella Typhimurium from liquid samples was developed and optimized. Both polyclonal and monoclonal antibodies have been used to couple with 1 micron sized......A very small number of bacterial pathogens may have fatal effects on food safety. In spite of having great advancements in bioanalytical methods, most of the accepted detection methods are still cultivation based and thus time consuming. This leads to an intense need for efficient and rapid methods...... and bacteria, immunocapture time, staining and buffering conditions for the viability assays were optimized. The capture efficiency of IMS was>98% for a range of Salmonella Typhimurium cell concentrations from 103 to 105/mL using 108/mL bead concentration. The method proved to have high (98%) specificity...

  6. Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans.

    Science.gov (United States)

    Catal, Mursel; King, Louis; Tumbalam, Pavani; Wiriyajitsomboon, Prissana; Kirk, William W; Adams, Gerard C

    2010-08-01

    A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates. Nuclear DNA content varies from 1.75x to 0.75x that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen.

  7. Hierarchical modeling for rare event detection and cell subset alignment across flow cytometry samples.

    Directory of Open Access Journals (Sweden)

    Andrew Cron

    Full Text Available Flow cytometry is the prototypical assay for multi-parameter single cell analysis, and is essential in vaccine and biomarker research for the enumeration of antigen-specific lymphocytes that are often found in extremely low frequencies (0.1% or less. Standard analysis of flow cytometry data relies on visual identification of cell subsets by experts, a process that is subjective and often difficult to reproduce. An alternative and more objective approach is the use of statistical models to identify cell subsets of interest in an automated fashion. Two specific challenges for automated analysis are to detect extremely low frequency event subsets without biasing the estimate by pre-processing enrichment, and the ability to align cell subsets across multiple data samples for comparative analysis. In this manuscript, we develop hierarchical modeling extensions to the Dirichlet Process Gaussian Mixture Model (DPGMM approach we have previously described for cell subset identification, and show that the hierarchical DPGMM (HDPGMM naturally generates an aligned data model that captures both commonalities and variations across multiple samples. HDPGMM also increases the sensitivity to extremely low frequency events by sharing information across multiple samples analyzed simultaneously. We validate the accuracy and reproducibility of HDPGMM estimates of antigen-specific T cells on clinically relevant reference peripheral blood mononuclear cell (PBMC samples with known frequencies of antigen-specific T cells. These cell samples take advantage of retrovirally TCR-transduced T cells spiked into autologous PBMC samples to give a defined number of antigen-specific T cells detectable by HLA-peptide multimer binding. We provide open source software that can take advantage of both multiple processors and GPU-acceleration to perform the numerically-demanding computations. We show that hierarchical modeling is a useful probabilistic approach that can provide a

  8. Label-free detection of circulating melanoma cells by in vivo photoacoustic flow cytometry

    Science.gov (United States)

    Wang, Xiaoling; Yang, Ping; Liu, Rongrong; Niu, Zhenyu; Suo, Yuanzhen; He, Hao; Gao, Wenyuan; Tang, Shuo; Wei, Xunbin

    2016-03-01

    Melanoma is a malignant tumor of melanocytes. Melanoma cells have high light absorption due to melanin highly contained in melanoma cells. This property is employed for the detection of circulating melanoma cell by in vivo photoacoustic flow cytometry (PAFC), which is based on photoacoustic effect. Compared to in vivo flow cytometry based on fluorescence, PAFC can employ high melanin content of melanoma cells as endogenous biomarkers to detect circulating melanoma cells in vivo. We have developed in vitro experiments to prove the ability of PAFC system of detecting photoacoustic signals from melanoma cells. For in vivo experiments, we have constructed a model of melanoma tumor bearing mice by inoculating highly metastatic murine melanoma cancer cells, B16F10 with subcutaneous injection. PA signals are detected in the blood vessels of mouse ears in vivo. The raw signal detected from target cells often contains some noise caused by electronic devices, such as background noise and thermal noise. We choose the Wavelet denoising method to effectively distinguish the target signal from background noise. Processing in time domain and frequency domain would be combined to analyze the signal after denoising. This algorithm contains time domain filter and frequency transformation. The frequency spectrum image of the signal contains distinctive features that can be used to analyze the property of target cells or particles. The processing methods have a great potential for analyzing signals accurately and rapidly. By counting circulating melanoma cells termly, we obtain the number variation of circulating melanoma cells as melanoma metastasized. Those results show that PAFC is a noninvasive and label-free method to detect melanoma metastases in blood or lymph circulation.

  9. Flow Cytometry: A New Approach for Indirect Assessment of Sperm Protamine Deficiency

    Directory of Open Access Journals (Sweden)

    Marziyeh Tavalaee

    2010-01-01

    Full Text Available Background: Flow cytometry (FCM has been extensively used to study mammalian sperm in theareas of clinical andrology and reproductive toxicology. FCM provides a powerful advantage overmicroscopy technique in terms of rapid, accurate and reproducible technology for the quantification ofvarious cell characteristics, including chromatin status. During spermiogenesis, histones are replacedby protamines resulting in a very condensed structure of sperm chromatin. Infertile men have anincreased sperm histone: protamine ratio than fertile counterparts. Chromomycin A3 (CMA3 stainingrepresents a useful tool for assessing the packaging quality of sperm chromatin and allows indirectvisualization of protamine deficiency. Routinely, fluorescence microscope is used for evaluation ofprotamine deficiency by CMA3. Considering the advantages of FCM and increasing use of CMA3 inassessment of protamine deficiency in the literature and its possible use as a diagnostic test, the aim ofthis study is to standardize this procedure for routine laboratory analysis.Materials and Methods: Semen samples were collected from 85 infertile men who referred toIsfahan Fertility and Infertility Center. A portion of semen sample was used for routine semenanalysis according to WHO criteria and the remainder were evaluated to standardize CMA3 stainingprocedure for fixation, the number of sperm and duration of exposure to CMA3. The results werecompared with standard fluorescent microscopic procedure. Percentage CMA3 positive sperm werecompared between flow cytometry and standard fluorescent microscopic procedure.Results: Our results show that fixation, the number of sperm and duration of exposure to CMA3can affect on FCM outcomes. In addition we show that the samples can be fixed, stained withCMA3, stores and then assessed for FCM.Conclusion: The optimal conditions for FCM assessment of CMA3 are: fixation, concentration of0.25 mg/ml, sperm density of 2 million/ml and exposure for 60

  10. Biomass measurement by flow cytometry during solid-state fermentation of basidiomycetes.

    Science.gov (United States)

    Steudler, Susanne; Böhmer, Ulrike; Weber, Jost; Bley, Thomas

    2015-02-01

    Solid-state fermentation (SSF) is a robust process that is well suited to the on-site cultivation of basidiomycetes that produce enzymes for the treatment of lignocellulosics. Reliable methods for biomass quantification are essential for the analysis of fungal growth kinetics. However, direct biomass determination is not possible during SSF because the fungi grow into the substrate and use it as a nutrient source. This necessitates the use of indirect methods that are either very laborious and time consuming or can only provide biomass measurements during certain growth periods. Here, we describe the development and optimization of a new rapid method for fungal biomass determination during SSF that is based on counting fungal nuclei by flow cytometry. Fungal biomass was grown on an organic substrate and its concentration was measured by isolating the nuclei from the fungal hyphae after cell disruption, staining them with SYTOX(®) Green, and then counting them using a flow cytometer. A calibration curve relating the dry biomass of the samples to their concentrations of nuclei was established. Multiple buffers and disruption methods were tested. The results obtained were compared with values determined using the method of ergosterol determination, a classical technique for fungal biomass measurement during SSF. Our new approach can be used to measure fungal biomass on a range of different scales, from 15 mL cultures to a laboratory reactor with a working volume of 10 L (developed by the Research Center for Medical Technology and Biotechnology (fzmb GmbH)). © 2014 International Society for Advancement of Cytometry.

  11. Gram-typing of mastitis bacteria in milk samples using flow cytometry.

    Science.gov (United States)

    Langerhuus, S N; Ingvartsen, K L; Bennedsgaard, T W; Røntved, C M

    2013-01-01

    Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identification of bacterial pathogens, although shipment of samples to diagnostic laboratories delays treatment decisions. Due to the lack of fast on-site tests that can identify the causative pathogens, antibiotic treatments are often initiated before bacterial identification. The present study describes a flow cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial cultures (4 gram-negative and 15 gram-positive strains) were analyzed, and biotin-conjugated wheat germ agglutinin and acridine orange florescence intensities were determined for gram-negative and gram-positive bacteria, respectively. Fluorescence cut-off values were established based on receiver operating characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1 and a specificity of 0.74, when classification was based on the previously established cut-off values. However, when receiver operating characteristic curves were constructed for the 53 mastitis cases, results indicate that a sensitivity and specificity of 1 could be reached if cut-off values were reduced. This flow cytometry-based technique could potentially provide dairy farmers and attending veterinarians with on-site information on bacterial gram-type and prevent ineffective

  12. Development of an Immunomagnetic Separation Method for Viable Salmonella Typhimurium Detected by Flow Cytometry

    DEFF Research Database (Denmark)

    Ahmed, Shakil; Rubahn, Horst-Günter; Erdmann, Helmut

    2016-01-01

    and bacteria, immunocapture time, staining and buffering conditions for the viability assays were optimized. The capture efficiency of IMS was>98% for a range of Salmonella Typhimurium cell concentrations from 103 to 105/mL using 108/mL bead concentration. The method proved to have high (98%) specificity...... paramagnetic particles for the preparation of immunomagnetic beads (IMBs). The most suitable antibody was chosen by applying an enzyme linked immunosorbent assay (ELISA), whereas living bacteria were detected by flow cytometry. The parameters for both IMS and flow cytometry e.g., concentration of bead...

  13. Hierarchical Bayesian mixture modelling for antigen-specific T-cell subtyping in combinatorially encoded flow cytometry studies

    DEFF Research Database (Denmark)

    Lin, Lin; Chan, Cliburn; Hadrup, Sine R

    2013-01-01

    Novel uses of automated flow cytometry technology for measuring levels of protein markers on thousands to millions of cells are promoting increasing need for relevant, customized Bayesian mixture modelling approaches in many areas of biomedical research and application. In studies of immune...... profiling in many biological areas, traditional flow cytometry measures relative levels of abundance of marker proteins using fluorescently labeled tags that identify specific markers by a single-color. One specific and important recent development in this area is the use of combinatorial marker assays...

  14. [Protein kinase C activation induces platelet apoptosis].

    Science.gov (United States)

    Zhao, Li-Li; Chen, Meng-Xing; Zhang, Ming-Yi; Dai, Ke-Sheng

    2013-10-01

    Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelets, or in the development of some platelet-related diseases. However, up to now, a little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase C (PKC) is highly expressed in platelets and plays central roles in regulating platelet functions. Although there is evidence indicating that PKC is involved in the regulation of apoptosis of nucleated cells, it is still unclear whether PKC plays a role in platelet apoptosis. The aim of this study was to investigate the role of PKC in platelet apoptosis. The effects of PKC on mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) exposure, and caspase-3 activation of platelets were analyzed by flow cytometry and Western blot. The results showed that the ΔΨm depolarization in platelets was induced by PKC activator in time-dependent manner, and the caspase-3 activation in platelets was induced by PKC in concentration-dependent manner. However, the platelets incubated with PKC inhibitor did not results in ΔΨm depolarization and PS exposure. It is concluded that the PKC activation induces platelet apoptosis through influencing the mitochondrial functions and activating caspase 3. The finds suggest a novel mechanism for PKC in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

  15. Detection, isolation, and capture of circulating breast cancer cells with photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran; Njoroge, Martin; Goldschmidt, Benjamin S.; Gaffigan, Brian; Rood, Kyle; Viator, John A.

    2013-03-01

    According to the CDC, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis, or the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems, significantly worsens the prognosis of any breast cancer patient. In this study, a technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser with a 5 ns pulse at 532 nm is used to interrogate thousands of cells with one pulse as they flow through the beam path. Cells which are pigmented, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to provide pigment. After which, the device is calibrated to demonstrate a single-cell detection limit. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25-45 breast cancer cells per 1 mL of blood. An in vitro photoacoustic flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy, it can also be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  16. Platelet adhesion from shear blood flow is controlled by near-wall rebounding collisions with erythrocytes.

    Science.gov (United States)

    Tokarev, A A; Butylin, A A; Ataullakhanov, F I

    2011-02-16

    The efficacy of platelet adhesion in shear flow is known to be substantially modulated by the physical presence of red blood cells (RBCs). The mechanisms of this regulation remain obscure due to the complicated character of platelet interactions with RBCs and vascular walls. To investigate this problem, we have created a mathematical model that takes into account shear-induced transport of platelets across the flow, platelet expulsion by the RBCs from the near-wall layer of the flow onto the wall, and reversible capture of platelets by the wall and their firm adhesion to it. This model analysis allowed us to obtain, for the first time to our knowledge, an analytical determination of the platelet adhesion rate constant as a function of the wall shear rate, hematocrit, and average sizes of platelets and RBCs. This formula provided a quantitative description of the results of previous in vitro adhesion experiments in perfusion chambers. The results of the simulations suggest that under a wide range of shear rates and hematocrit values, the rate of platelet adhesion from the blood flow is mainly limited by the frequency of their near-wall rebounding collisions with RBCs. This finding reveals the mechanism by which erythrocytes physically control platelet hemostasis.

  17. Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

    Directory of Open Access Journals (Sweden)

    Keith B Neeves

    Full Text Available Microfluidic flow assays (MFA that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T, the rate of platelet accumulation (V(PLT, and platelet surface coverage (SC. A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT and SC at all wall shear rates. A longer Lag(T for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104 and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

  18. Inertial microfluidics for sheath-less high-throughput flow cytometry.

    Science.gov (United States)

    Bhagat, Ali Asgar S; Kuntaegowdanahalli, Sathyakumar S; Kaval, Necati; Seliskar, Carl J; Papautsky, Ian

    2010-04-01

    Flow cytometer is a powerful single cell analysis tool that allows multi-parametric study of suspended cells. Most commercial flow cytometers available today are bulky, expensive instruments requiring high maintenance costs and specially trained personnel for operation. Hence, there is a need to develop a low cost, portable alternative that will aid in making this powerful research tool more accessible. In this paper we describe a sheath-less, on-chip flow cytometry system based on the principle of Dean coupled inertial microfluidics. The design takes advantage of the Dean drag and inertial lift forces acting on particles flowing through a spiral microchannel to focus them in 3-D at a single position across the microchannel cross-section. Unlike the previously reported micro-flow cytometers, the developed system relies entirely on the microchannel geometry for particle focusing, eliminating the need for complex microchannel designs and additional microfluidic plumbing associated with sheath-based techniques. In this work, a 10-loop spiral microchannel 100 microm wide and 50 microm high was used to focus 6 microm particles in 3-D. The focused particle stream was detected with a laser induced fluorescence (LIF) setup. The microfluidic system was shown to have a high throughput of 2,100 particles/sec. Finally, the viability of the developed technique for cell counting was demonstrated using SH-SY5Y neuroblastoma cells. The passive focusing principle and the planar nature of the described design will permit easy integration with existing lab-on-a-chip (LOC) systems.

  19. Immunophenotype Discovery, Hierarchical Organization, and Template-based Classification of Flow Cytometry Samples

    Directory of Open Access Journals (Sweden)

    Ariful Azad

    2016-08-01

    Full Text Available We describe algorithms for discovering immunophenotypes from large collections of flow cytometry (FC samples, and using them to organize the samples into a hierarchy based on phenotypic similarity. The hierarchical organization is helpful for effective and robust cytometry data mining, including the creation of collections of cell populations characteristic of different classes of samples, robust classification, and anomaly detection. We summarize a set of samples belonging to a biological class or category with a statistically derived template for the class. Whereas individual samples are represented in terms of their cell populations (clusters, a template consists of generic meta-populations (a group of homogeneous cell populations obtained from the samples in a class that describe key phenotypes shared among all those samples. We organize an FC data collection in a hierarchical data structure that supports the identification of immunophenotypes relevant to clinical diagnosis. A robust template-based classification scheme is also developed, but our primary focus is in the discovery of phenotypic signatures and inter-sample relationships in an FC data collection. This collective analysis approach is more efficient and robust since templates describe phenotypic signatures common to cell populations in several samples, while ignoring noise and small sample-specific variations.We have applied the template-base scheme to analyze several data setsincluding one representing a healthy immune system, and one of Acute Myeloid Leukemia (AMLsamples. The last task is challenging due to the phenotypic heterogeneity of the severalsubtypes of AML. However, we identified thirteen immunophenotypes corresponding to subtypes of AML, and were able to distinguish Acute Promyelocytic Leukemia from other subtypes of AML.

  20. Flow cytometry immunophenotypic analysis of Philadelphia-negative myeloproliferative neoplasms: Correlation with histopathologic features.

    Science.gov (United States)

    Ouyang, Juan; Zheng, Wenli; Shen, Qi; Goswami, Maitrayee; Jorgensen, Jeffrey L; Medeiros, L Jeffrey; Wang, S A

    2015-01-01

    Compared with the proven utility of flow cytometry immunophenotyping (FCI) analysis in the workup of myelodysplastic syndromes (MDS), immunophenotypic alterations in myeloproliferative neoplasms (MPN) have been less studied and the potential utility of FCI is not defined. Bone marrow (BM) samples of 83 Philadelphia-negative MPN patients were assessed by multicolor FCI including 27 with essential thrombocythemia (ET); 17 polycythemia vera (PV); 33 primary myelofibrosis (PMF) and 6 MPN-unclassifiable (MPN-U). The time interval from initial diagnosis of MPN to FCI analysis was 18 months (0-370). Ninety-five age-matched MDS patients with a similar BM blast count were included for comparison. Immunophenotypic alterations, either in CD34(+) cells or myelomonocytic cells, were detected in 82 of 83 (99%) MPN cases. FCI abnormalities were more frequently observed in cases with substantial myelofibrosis but not different between PMF and fibrotic stage of ET/PV. Furthermore, FCI abnormalities were more frequent in cases with ≥5% BM blasts and/or circulating blasts (P = 0.006); as well as cases with an abnormal karyotype (P = 0.036); but not associated with morphologic dysplasia or JAK2 mutation status. Comparing with MDS, FCI abnormalities were overall less pronounced in MPN cases (P = 0.001). MPNs exhibit frequent immunophenotypic alterations, more pronounced in cases with adverse histopathologic features. These findings illustrate that immunophenotypic alterations are a part of constellational findings in MPN, and correlate progressively with disease stage. The study results also suggest a role of FCI in diagnosis of MPN and monitoring disease over time and after therapy. © 2014 International Clinical Cytometry Society.

  1. Femtosecond laser fabrication of fiber based optofluidic platform for flow cytometry applications

    Science.gov (United States)

    Serhatlioglu, Murat; Elbuken, Caglar; Ortac, Bulend; Solmaz, Mehmet E.

    2017-02-01

    Miniaturized optofluidic platforms play an important role in bio-analysis, detection and diagnostic applications. The advantages of such miniaturized devices are extremely low sample requirement, low cost development and rapid analysis capabilities. Fused silica is advantageous for optofluidic systems due to properties such as being chemically inert, mechanically stable, and optically transparent to a wide spectrum of light. As a three dimensional manufacturing method, femtosecond laser scanning followed by chemical etching shows great potential to fabricate glass based optofluidic chips. In this study, we demonstrate fabrication of all-fiber based, optofluidic flow cytometer in fused silica glass by femtosecond laser machining. 3D particle focusing was achieved through a straightforward planar chip design with two separately fabricated fused silica glass slides thermally bonded together. Bioparticles in a fluid stream encounter with optical interrogation region specifically designed to allocate 405nm single mode fiber laser source and two multi-mode collection fibers for forward scattering (FSC) and side scattering (SSC) signals detection. Detected signal data collected with oscilloscope and post processed with MATLAB script file. We were able to count number of events over 4000events/sec, and achieve size distribution for 5.95μm monodisperse polystyrene beads using FSC and SSC signals. Our platform shows promise for optical and fluidic miniaturization of flow cytometry systems.

  2. Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

    Institute of Scientific and Technical Information of China (English)

    LI ZHU; JUN-PENG CAI; QING CHEN; SHOU-YI YU

    2007-01-01

    Objective To develop an in situ PCR in combination with flow cytometry (ISPCR-FCM) for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene (ctxAB gene). Cells subjected to ISPCR showed the presences of ctxAB gene both in epifiuorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low (105 cells/mL), while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

  3. Overestimation of heterotrophic bacteria in the Sargasso Sea: direct evidence by flow and imaging cytometry

    Science.gov (United States)

    Sieracki, Michael E.; Haugen, Elin M.; Cucci, Terry L.

    1995-08-01

    Accurate measurements of bacterial biomass in the ocean are needed for modeling marine microbial food webs and global biogeochemical cycling. We present direct evidence that previous estimates of heterotrophic bacteria biomass in the oligotrophic ocean are confounded by the presence of the abundant photosynthetic procaryote, Prochlorococcus. The chlorophyll autofluorescence of these photosynthetic bacterial cells is very faint and fades rapidly under epifluorescence microscopy. Detection and enumeration of these cells thus far has almost exclusively been by flow cytometry. Using a cooled, charge-coupled device (CCD) camera we were able to image these cells for direct biovolume measurements. A double-exposed image of DAPI-stained Prochlorococcus cells shows that they are indistinguishable from heterotrophic bacteria in standard slide preparations. At two Sargasso Sea stations Prochlorococcus could cause an overestimation of surface (top 150 m) integrated heterotrophic bacterial biovolume (biomass) of 18 and 22% determined by standard microscope methods. At the subsurface chlorophyll maximum Prochlorococcus was 33 and 43% of the heterotrophic bacterial biovolume (biomass) at these stations. Prochlorococcus cell size increased from 0.05 μm 3 in the surface mixed layer to about 0.2 μm 3 below 100 m, confirming previous interpretations of flow cytometric light scatter measurements. Shifting biomass from the heterotrophic bacteria pool to the primary producer compartment has significant implications for ecosystem structure and trophic transfer in marine food webs.

  4. Evaluation of a new flow cytometry crossmatch procedure for simultaneous detection of cytotoxicity and antibody binding.

    Science.gov (United States)

    Alheim, M; Paul, P K; Hauzenberger, D-M; Wikström, A-C

    2013-08-01

    In this study we have evaluated an alternative 96-well format flow cytometry based (FCtox) method which enable simultaneous detection of cytotoxicity and human leukocyte antigen (HLA) antibody binding. Comparable results were obtained in side-by-side comparisons with conventional complement-dependent cytotoxicity (CDC) and flow cytometric crossmatch (FCXM) in terms of sensitivity and specificity. There was 91 and 93% agreement between results obtained by FCtox and CDC for T and B cells, respectively. In addition, comparable results were obtained with FCtox IgG and FCXM IgG for both T and B cells. Furthermore, compared with a recently developed and highly sensitive Luminex based C1q assay we obtained close to 90% method agreement with the FCtox assay. Our alternative cytotoxicity and IgG binding assay which exhibit low intra-and inter-assay variation will improve the workflow and speed up the pre-transplant testing and also allow continuous monitoring of assay performance and proper quality assurance. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Selective grazing by adults and larvae of the zebra mussel (Dreissena polymorpha): application of flow cytometry to natural seston

    NARCIS (Netherlands)

    Pires, L.M.D.; Jonker, R.R.; Donk, E. van; Laanbroek, H.J.

    2004-01-01

    1. Selective grazing of adults and larvae of the zebra mussel (Dreissena polymorpha) on phytoplankton and detritus from both laboratory cultures and natural seston was quantified using flow cytometry. 2. Mean clearance rate of adult zebra mussels was higher on a mixture of the green alga Scenedesmus

  6. Flow cytometry with gold nanoparticlesand their clusters as scattering contrast agents: FDTD simulation of light-cell interaction

    DEFF Research Database (Denmark)

    Tanev, Stoyan; Sun, Wenbo; Pond, James

    2009-01-01

    The formulation of the Finite-Difference Time-Domain (FDTD) approach is presented in the framework of its potential applications to in vivo flow cytometry based on light scattering. The consideration is focused on comparison of light scattering by a single biological cell alone in controlled refr...

  7. Flow cytometry total cell counts: a field study assessing microbiological water quality and growth in unchlorinated drinking water distribution systems

    NARCIS (Netherlands)

    Liu, G.; Van der Mark, E.J.; Verberk, J.Q.; Van Dijk, J.C.

    2013-01-01

    e objective of this study was to evaluate the application of flow cytometry total cell counts (TCCs) as a parameter to assess microbial growth in drinking water distribution systems and to determine the relationships between different parameters describing the biostability of treated water. A one-ye

  8. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress.

    Science.gov (United States)

    Amor, Kaouther Ben; Breeuwer, Pieter; Verbaarschot, Patrick; Rombouts, Frank M; Akkermans, Antoon D L; De Vos, Willem M; Abee, Tjakko

    2002-11-01

    Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strains during stress exposure. However, the flow cytometry results tended to overestimate the viability of the two strains compared to plate counts, which appeared to be related to the nonculturability of a fraction of the population as a result of sublethal injury caused by bile salts. When the cells were simultaneously stained with cFDA and PI, flow cytometry and cell sorting revealed a striking physiological heterogeneity within the stressed bifidobacterium population. Three subpopulations could be identified based on their differential uptake of the probes: cF-stained, cF and PI double-stained, and PI-stained subpopulations, representing viable, injured, and dead cells, respectively. Following sorting and recovery, a significant fraction of the double-stained subpopulation (40%) could resume growth on agar plates. Our results show that in situ assessment of the physiological activity of stressed bifidobacteria using multiparameter flow cytometry and cell sorting may provide a powerful and sensitive tool for assessment of the viability and stability of probiotics.

  9. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead bifidobacterium cells during bile salt stress

    NARCIS (Netherlands)

    Ben Amor, K.; Breeuwer, P.; Verbaarschot, P.; Rombouts, F.M.; Akkermans, A.D.L.; Vos, de W.M.; Abee, T.

    2002-01-01

    Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activi

  10. Comparison of real time RT-PCR and flow cytometry methods for evaluation of biological activity of recombinant human erythropoietin

    Directory of Open Access Journals (Sweden)

    Sepehrizadeh Z

    2008-05-01

    Full Text Available Background: Evaluation of bioactivity of recombinant erythropoietin is essential for pharmaceutical industry, quality control authorities and researchers. The purpose of this study was to compare real time RT-PCR and flow cytometry for the assay of biological activity of recombinant erythropoietin. Methods: Three concentrations of recombinant erythropoietin BRP (80, 40 and 20 IU/ml were injected subcutaneously to mice. After 4 days the blood was collected and used for reticulocyte counts by flow cytometry and also for the RNA extraction. Real time RT-PCR amplification was carried out for β-globin. Results and conclusion: There was a significant correlation between the total RNA amounts (R2= 0.9995, relative quantity of β-globin mRNA (R2= 0.984 and reticulocyte counts (R2= 0.9742 with rhEpo concentrations. Total RNA and quantitative RT-PCR showed significant dose dependent results as well the reticulocyte counts by flow cytometry for the biological activity assay of rhEpo and so these methods could be considered as alternatives for flow cytometry.

  11. Utilization of flow cytometry to identify chimeral sectors in leaf tissue of Lolium multiflorum x L. arundinaceum hybrids

    Science.gov (United States)

    We have identified a method whereby Lolium multiflorum (Lm) or L. arundinaceum (Fa) genomes are preferentially eliminated through a mitotic loss behavior in interspecific Lm x Fa F1 hybrids, generating either dihaploid Lm lines or Fa lines. Flow cytometry, a method for rapidly characterizing optical...

  12. Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry

    NARCIS (Netherlands)

    Terstappen, Leon W.M.M.; Johnsen, Steen; Segers-Nolten, Ine M.J.; Loken, Michael R.

    1990-01-01

    The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma

  13. Automation of a phospho-STAT5 staining procedure for flow cytometry for application in drug discovery

    NARCIS (Netherlands)

    Malergue, Fabrice; van Agthoven, Andreas; Scifo, Caroline; Egan, Dave; Strous, Ger J

    2015-01-01

    Drug discovery often requires the screening of compound libraries on tissue cultured cells. Some major targets in drug discovery belong to signal transduction pathways, and PerFix EXPOSE* allows easy flow cytometry phospho assays. We thus investigated the possibility to further simplify and automate

  14. Flow cytometry total cell counts: a field study assessing microbiological water quality and growth in unchlorinated drinking water distribution systems

    NARCIS (Netherlands)

    Liu, G.; Van der Mark, E.J.; Verberk, J.Q.; Van Dijk, J.C.

    2013-01-01

    e objective of this study was to evaluate the application of flow cytometry total cell counts (TCCs) as a parameter to assess microbial growth in drinking water distribution systems and to determine the relationships between different parameters describing the biostability of treated water. A

  15. Where do the platelets go? A simulation study of fully resolved blood flow through aneurysmal vessels

    NARCIS (Netherlands)

    Mountrakis, L.; Lorenz, E.; Hoekstra, A.G.

    2013-01-01

    Despite the importance of platelets in the formation of a thrombus, their transport in complex flows has not yet been studied in detail. In this paper we simulated red blood cells and platelets to explore their transport behaviour in aneurysmal geometries. We considered two aneurysms with different

  16. Analysis of basophil activation by flow cytometry in pediatric house dust mite allergy.

    Science.gov (United States)

    González-Muñoz, Miguel; Villota, Julian; Moneo, Ignacio

    2008-06-01

    Detection of allergen-induced basophil activation by flow cytometry has been shown to be a useful tool for allergy diagnosis. The aim of this study was to assess the potential of this technique for the diagnosis of pediatric house dust mite allergy. Quantification of total and specific IgE and basophil activation test were performed to evaluate mite allergic (n = 24), atopic (n = 23), and non-allergic children (n = 9). Allergen-induced basophil activation was detected as a CD63-upregulation. Receiver operating characteristics (ROC) curve analysis was performed to calculate the optimal cut-off value of activated basophils discriminating mite allergic and non-allergic children. ROC curve analysis yielded a threshold value of 18% activated basophils when mite-sensitized and atopic children were studied [area under the curve (AUC) = 0.99, 95% confidence interval (CI) = 0.97-1.01, p Analysis of the data obtained with 1.6 microg/ml mite extract defined a cut-off value of 8% activated basophils (AUC = 0.96, 95% CI = 0.91-1.01; p basophils (AUC = 1.0) with 16 microg/ml allergen extract and a sensitivity and specificity of 100%. The same threshold and specificity values were obtained with 1.6 microg/ml extract (AUC = 97%, 95% CI = 0.92-1.02; p basophil activation tests and high specific IgE (>43 kU/l) values for Dermatophagoides pteronyssinus allergen. They also showed positive prick (wheal diameter >1.0 cm) and basophil activation (>87%) tests and high specific IgE (>100 kU/l) with shrimp allergen. Shrimp sensitization was demonstrated by high levels of Pen a 1-specific IgE (>100 kU/l). Cross-reactivity between mite and shrimp was confirmed by fluorescence enzyme immunoassay (FEIA-CAP) inhibition study in these two cases. This study demonstrated that the analysis of allergen-induced CD63 upregulation by flow cytometry is a reliable tool for diagnosis of mite allergy in pediatric patients, with sensitivity similar to routine diagnostic tests and a higher specificity

  17. Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma

    Directory of Open Access Journals (Sweden)

    Blix Egil S

    2012-10-01

    Full Text Available Abstract Background Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL and marginal zone lymphoma (MZL patients. Methods Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR, CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling. Results Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB, p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells. Conclusions BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation

  18. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    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

  19. Matrix metalloproteinase-2 enhances platelet deposition on collagen under flow conditions.

    Science.gov (United States)

    Guglielmini, Giuseppe; Appolloni, Viviana; Momi, Stefania; De Groot, Philip G; Battiston, Monica; De Marco, Luigi; Falcinelli, Emanuela; Gresele, Paolo

    2016-01-01

    Platelets contain and release matrix metalloproteinase-2 (MMP-2) that in turn potentiates platelet aggregation. Platelet deposition on a damaged vascular wall is the first, crucial, step leading to thrombosis. Little is known about the effects of MMP-2 on platelet activation and adhesion under flow conditions. We studied the effect of MMP-2 on shear-dependent platelet activation using the O'Brien filtration system, and on platelet deposition using a parallel-plate perfusion chamber. Preincubation of human whole blood with active MMP-2 (50 ng/ml, i.e. 0.78 nM) shortened filter closure time (from 51.8 ± 3.6 sec to 40 ± 2.7 sec, pMMP-2 inhibitor. High shear stress induced the release of MMP-2 from platelets, while TIMP-2 levels were not significantly reduced, therefore, the MMP-2/TIMP-2 ratio increased significantly showing enhanced MMP-2 activity. Preincubation of whole blood with active MMP-2 (0.5 to 50 ng/ml, i.e 0.0078 to 0.78 nM) increased dose-dependently human platelet deposition on collagen under high shear-rate flow conditions (3000 sec⁻¹) (maximum +47.0 ± 11.9%, pMMP-2 inhibitor reduced platelet deposition. In real-time microscopy studies, increased deposition of platelets on collagen induced by MMP-2 started 85 sec from the beginning of perfusion, and was abolished by a GPIIb/IIIa antagonist, while MMP-2 had no effect on platelet deposition on fibrinogen or VWF. Confocal microscopy showed that MMP-2 enhances thrombus volume (+20.0 ± 3.0% vs control) rather than adhesion. In conclusion, we show that MMP-2 potentiates shear-induced platelet activation by enhancing thrombus formation.

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

    Science.gov (United States)

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

    2006-11-01

    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.

  1. Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry.

    Science.gov (United States)

    Saint-Ruf, Claude; Crussard, Steve; Franceschi, Christine; Orenga, Sylvain; Ouattara, Jasmine; Ramjeet, Mahendrasingh; Surre, Jérémy; Matic, Ivan

    2016-01-01

    Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.

  2. In-vitro human spermatozoa nuclear decondensation assessed by flow cytometry.

    Science.gov (United States)

    Samocha-Bone, D; Lewin, L M; Weissenberg, R; Madgar, Y; Soffer, Y; Shochat, L; Golan, R

    1998-02-01

    The process of sperm chromatin decondensation occurs when a spermatozoon enters an ovum. Protamine disulphide bonds are reduced to SH and the polycationic protamines combine with the polyanionic egg protein, nucleoplasmin, thus being stripped from DNA which then combines with histones. Defective chromatin decondensation will thus prevent further development of the male pronucleus. In this study human sperm samples were incubated in vitro at 28 degrees C (using a medium in which the polyanion, heparin, substitutes for nucleoplasmin and beta-mercaptoethanol for egg glutathione) for 10, 20 and 30 min before stopping the reaction with formalin (to 3.6%). The DNA of the fixed cells was stained with Acridine Orange by a one-step method and subjected to flow cytometry and data analysis, in which a zone characteristic of condensed chromatin is outlined on red-green fluorescence contour plots. After 20 min of incubation 97% of the control spermatozoa that were in the mature window (WIN M) had decondensed and moved out of this region. Defects in sperm decondensation were seen in four semen samples of the 20 that were tested. In cases where spermatozoa fail to produce a fertilized egg the cause may lie with defective chromatin quality, including failure of the sperm chromatin to decondense. The method described here is a simple procedure for detecting sperm samples containing such defective cells.

  3. Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo

    Science.gov (United States)

    Nedosekin, Dmitry A.; Sarimollaoglu, Mustafa; Proskurnin, Mikhail A.

    2016-01-01

    Control of sickle cell disease (SCD) stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic (PA) flow cytometry (PAFC) has a potential for real-time monitoring of circulating sickled cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT) and PA spectral imaging of sickle red blood cells (sRBCs) expressing SCD-associated hemoglobin (HbS) compared to normal red blood cells (nRBCs). We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2–4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial hemoglobin heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode) as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs both in vitro and in vivo. PMID:27699143

  4. Use of Flow Cytometry To Follow the Physiological States of Microorganisms in Cider Fermentation Processes

    Science.gov (United States)

    Herrero, Mónica; Quirós, Covadonga; García, Luis A.; Díaz, Mario

    2006-01-01

    The flow cytometry (FC) technique used with certain fluorescent dyes (ChemChrome V6 [CV6], DRAQ5, and PI) has proven useful to label and to detect different physiological states of yeast and malolactic bacterium starters conducting cider fermentation over time (by performing sequential inoculation of microorganisms). First, the technique was tested with pure cultures of both types of microorganisms grown in synthetic media under different induced stress conditions. Metabolically active cells detected by FC and by the standard plate-counting method for both types of microorganisms in fresh overnight pure cultures gave good correlations between the two techniques in samples taken at this stage. Otherwise, combining the results obtained by FC and plating during alcoholic and malolactic fermentation over time in the cider-making process, different subpopulations were detected, showing significant differences between the methods. A small number of studies have applied the FC technique to analyze fermentation processes and mixed cultures over time. The results were used to postulate equations explaining the different physiological states in cell populations taken from fresh, pure overnight cultures under nonstress conditions or cells subjected to stress conditions over time, either under a pure-culture fermentation process (in this work, corresponding to alcoholic fermentation) or under mixed-fermentation conditions (for the malolactic-fermentation phase), that could be useful to improve the control of the processes. PMID:17021224

  5. Studying the numeration methods of signals with unstable background for in vivo flow cytometry

    Science.gov (United States)

    Wang, Xiaoling; Suo, Yuanzhen; Wei, Dan; He, Hao; Wei, Xunbin

    2016-10-01

    In recent years, the in vivo flow cytometry (IVFC) has been a useful technology in detecting and quantifying the circulating cells dynamically in living animals, especially in the research related to the cell tracking and the cancer metastasis. In practice, however, the unstable background signals caused by the experiment animals' respiratory movement, limb movement and photo-bleaching of tissues' auto-fluorescence exist in many IVFC data, which could affect the accuracy of cell counting results in the following post-processing procedure, making the IVFC signals less available. Here we developed a signal processing method that could effectively correct the unstable background signals by using methods combining interpolating, fitting, automatic segmenting and wavelet-based denoising. Compared with the previously used non-correction methods, i.e., the "line-gating" method or the automatic threshold method, this method showed a higher accuracy and efficiency in counting cell numbers of IVFC signals, as well as demonstrating a better statistic results in the Pearson's correlation coefficient R2 and the mean-squared error (MSE).

  6. Characterization of type II alveolar epithelial cells by flow cytometry and fluorescent markers.

    Science.gov (United States)

    Rochat, T R; Casale, J M; Hunninghake, G W

    1988-10-01

    Type II alveolar epithelial cells play a crucial role in maintaining the structure and functions of pulmonary alveoli. A number of techniques have been described to isolate type II cells for in vitro studies; however, type II cell suspensions isolated by each technique are still contaminated by macrophages or monocytes. The present studies describe the use of flow cytometry to accurately characterize the composition of these cell suspensions. With freshly isolated type II cell suspensions, type II cells could be distinguished from macrophages and monocytes by two methods: (1) the combination of natural fluorescence and orthogonal light scatter, or (2) the use of monoclonal antibodies OX-1 (directed against a common leukocyte antigen present on rat macrophages and monocytes) and PKK-1 (directed against cytokeratin intermediate filaments present in type II cells). With cultured type II cells, the combination of natural fluorescence and orthogonal light scatter did not distinguish between type II cells and macrophages or monocytes; however, the monoclonal antibodies OX-I and PKK-1 continued to distinguish between these cell types. As an example of the use of these techniques, the methods were used to define the sequential expression of class I and II major histocompatibility antigens on both type II cells and on macrophages or monocytes in the same cell preparations. These methods are of potential value in isolating pure populations either of type II cells or of macrophages or monocytes by cell sorting and in accurately identifying the cells present in type II cell suspensions or cultures.

  7. Seminal plasma applied post-thawing affects boar sperm physiology: a flow cytometry study.

    Science.gov (United States)

    Fernández-Gago, Rocío; Domínguez, Juan Carlos; Martínez-Pastor, Felipe

    2013-09-01

    Cryopreservation induces extensive biophysical and biochemical changes in the sperm. In the present study, we used flow cytometry to assess the capacitation-like status of frozen-thawed boar spermatozoa and its relationship with intracellular calcium, assessment of membrane fluidity, modification of thiol groups in plasma membrane proteins, reactive oxygen species (ROS) levels, viability, acrosomal status, and mitochondrial activity. This experiment was performed to verify the effect of adding seminal plasma on post-thaw sperm functions. To determine these effects after cryopreservation, frozen-thawed semen from seven boars was examined after supplementation with different concentrations of pooled seminal plasma (0%, 10%, and 50%) at various times of incubation from 0 to 4 hours. Incubation caused a decrease in membrane integrity and an increase in acrosomal damage, with small changes in other parameters (P > 0.05). Although 10% seminal plasma showed few differences with 0% (ROS increase at 4 hours, P boar spermatozoa, possibly through membrane changes and ROS increase. Although some effects were detrimental, the stimulatory effect of 50% seminal plasma could favor the performance of post-thawed boar semen, as showed in the field (García JC, Domínguez JC, Peña FJ, Alegre B, Gonzalez R, Castro MJ, Habing GG, Kirkwood RN. Thawing boar semen in the presence of seminal plasma: effects on sperm quality and fertility. Anim Reprod Sci 2010;119:160-5).

  8. Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Chengzhong Cai

    2016-01-01

    Full Text Available Control of sickle cell disease (SCD stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic flow cytometry (PAFC has a potential for real-time monitoring of circulating sickle cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT and PA spectral imaging of sickle red blood cells (sRBCs expressing SCD-associated hemoglobin (HbS compared to normal red blood cells (nRBCs. We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2–4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial Hb heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs in vitro and in vivo.

  9. Duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes.

    Science.gov (United States)

    Young, Susan M; Bologa, Cristian M; Fara, Dan; Bryant, Bj K; Strouse, Juan Jacob; Arterburn, Jeffrey B; Ye, Richard D; Oprea, Tudor I; Prossnitz, Eric R; Sklar, Larry A; Edwards, Bruce S

    2009-03-01

    Of recent, clinical interest have been two related human G-protein coupled receptors: formylpeptide receptor (FPR), linked to antibacterial inflammation and malignant glioma cell metastasis; and FPR like-1 (FPRL1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, and prion diseases. In association with the National Institutes of Health (NIH) Molecular Library Screening Network, we implemented a flow-cytometry-based high-throughput screening (HTS) approach for identifying selective small molecule FPR and FPRL1 ligands. The screening assay measured the ability of test compounds to competitively displace a high-affinity, fluorescein- labeled peptide ligand from FPR, FPRL1, or both. U937 cells expressing FPR and rat basophil leukemia (RBL) cells expressing FPRL1 were tested together in a "duplex" format. The U937 cells were color coded with red-fluorescent dye allowing their distinction during analysis. Compounds, cells, and fluorescent ligand were sequentially combined (no wash) in 15 microl assay volumes in 384-well plates. Throughput averaged approximately 11 min per plate to analyze approximately 4,000 cells ( approximately 2,000/receptor) in a 2 microl aspirate from each well. In primary single concentration HTS of 24,304 NIH Small Molecule Repository compounds, 253 resulted in inhibition >30% (181 for FPR, 72 for FPRL1) of which 40 had selective binding inhibition constants (K(i)) probes.

  10. Analysis of bacterial-surface-specific antibodies in body fluids using bacterial flow cytometry.

    Science.gov (United States)

    Moor, Kathrin; Fadlallah, Jehane; Toska, Albulena; Sterlin, Delphine; Balmer, Maria L; Macpherson, Andrew J; Gorochov, Guy; Larsen, Martin; Slack, Emma

    2016-08-01

    Antibacterial antibody responses that target surfaces of live bacteria or secreted toxins are likely to be relevant in controlling bacterial pathogenesis. The ability to specifically quantify bacterial-surface-binding antibodies is therefore highly attractive as a quantitative correlate of immune protection. Here, binding of antibodies from various body fluids to pure-cultured live bacteria is made visible with fluorophore-conjugated secondary antibodies and measured by flow cytometry. We indicate the necessary controls for excluding nonspecific binding and also demonstrate a cross-adsorption technique for determining the extent of cross-reactivity. This technique has numerous advantages over standard ELISA and western blotting techniques because of its independence from scaffold binding, exclusion of cross-reactive elements from lysed bacteria and ability to visualize bacterial subpopulations. In addition, less than 10(5) bacteria and less than 10 μg of antibody are required per sample. The technique requires 3-4 h of hands-on experimentation and analysis. Moreover, it can be combined with automation and mutliplexing for high-throughput applications.

  11. Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines.

    Directory of Open Access Journals (Sweden)

    Steven G Smith

    Full Text Available Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

  12. Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

    Science.gov (United States)

    Yason, John Anthony; Tan, Kevin Shyong Wei

    2015-01-01

    Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

  13. Immune Monitoring in Cancer Vaccine Clinical Trials: Critical Issues of Functional Flow Cytometry-Based Assays

    Directory of Open Access Journals (Sweden)

    Iole Macchia

    2013-01-01

    Full Text Available 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 cytometry- (FCM- based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

  14. Genome size of Alexandrium catenella and Gracilariopsis lemaneiformis estimated by flow cytometry

    Science.gov (United States)

    Du, Qingwei; Sui, Zhenghong; Chang, Lianpeng; Wei, Huihui; Liu, Yuan; Mi, Ping; Shang, Erlei; Zeeshan, Niaz; Que, Zhou

    2016-08-01

    Flow cytometry (FCM) technique has been widely applied to estimating the genome size of various higher plants. However, there is few report about its application in algae. In this study, an optimized procedure of FCM was exploited to estimate the genome size of two eukaryotic algae. For analyzing Alexandrium catenella, an important red tide species, the whole cell instead of isolated nucleus was studied, and chicken erythrocytes were used as an internal reference. The genome size of A. catenella was estimated to be 56.48 ± 4.14 Gb (1C), approximately nineteen times larger than that of human genome. For analyzing Gracilariopsis lemaneiformis, an important economical red alga, the purified nucleus was employed, and Arabidopsis thaliana and Chondrus crispus were used as internal references, respectively. The genome size of Gp. lemaneiformis was 97.35 ± 2.58 Mb (1C) and 112.73 ± 14.00 Mb (1C), respectively, depending on the different internal references. The results of this research will promote the related studies on the genomics and evolution of these two species.

  15. Flow cytometry analysis of cancer cell death induced by the extract of Thai plant Ellipeiopsis cherrevensis.

    Science.gov (United States)

    Yumoto, Ryoko; Kakizoe, Saki; Nagai, Junya; Patanasethanont, Denpong; Sripanidkulchai, Bung-Orn; Takano, Mikihisa

    2013-01-01

      The mechanism of cancer cell death induced by KP018, an ethanol extract of the Thai plant Ellipeiopsis cherrevensis, was examined in paclitaxel-resistant HepG2 (PR-HepG2) and colon-26 cells using flow cytometry. In PR-HepG2 cells, KP018 induced necrosis in a concentration-dependent manner. Necrosis of PR-HepG2 cells induced by KP018 as well as by hydrogen peroxide was suppressed by co-treatment of the cells with N-acetylcysteine. KP018 decreased the viability of colon-26 cells with an IC50 value of 15.1 µg/mL, which was estimated by XTT assay. As observed in PR-HepG2 cells, KP018 induced necrosis and the necrosis was suppressed by N-acetylcysteine in colon-26 cells. In addition, using colon-26 solid tumor-bearing mice, KP018 was found to suppress tumor growth without apparent toxicities under in vivo conditions. These results indicate that KP018 induces necrosis rather than apoptosis in these cancer cells, and reactive oxygen species such as hydrogen peroxide would be involved in KP018-induced necrosis. KP018 may be a useful source to search for a new anticancer drug that can be used for the chemotherapy of multidrug-resistant tumors.

  16. Utility of flow cytometry studies in the management of patients with multiple myeloma.

    Science.gov (United States)

    Paiva, Bruno; Merino, Juana; San Miguel, Jesús F

    2016-11-01

    Although the input of multiparameter flow cytometry (MFC) into the clinical management of multiple myeloma patients has faced some reluctance, continuously growing evidence supports the utility of MFC in this disease. MFC immunophenotyping of bone marrow and peripheral blood plasma cells affords cost-effective assessment of clonality, and provides prognostic information on the risk of progression in smoldering multiple myeloma, and the identification of active multiple myeloma patients with dismal outcome (e.g., high numbers of circulating tumor cells) or long-term survival despite suboptimal responses through the characterization of monoclonal gammopathy of undetermined significance-like phenotypes. Extensive data indicate that minimal residual disease (MRD) monitoring can be used as biomarker to evaluate treatment efficacy and act as surrogate for survival. The time has come to address within clinical trials the exact role of baseline risk factors and MRD monitoring for tailored therapy in multiple myeloma, which implies systematic usage of highly sensitive cost-effective, readily available, and standardized MRD techniques such as MFC. Next-generation MFC should be considered mandatory in the routine evaluation of multiple myeloma patients both at diagnosis and after therapy, and represents an attractive technique to integrate with high-throughput DNA and RNA-seq methods to help in understanding the mechanisms behind dissemination and chemoresistance of multiple myeloma.

  17. Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.

    Science.gov (United States)

    Manti, Anita; Boi, Paola; Amalfitano, Stefano; Puddu, Alberto; Papa, Stefano

    2011-12-01

    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.

  18. Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry.

    Science.gov (United States)

    Beaton-Green, Lindsay A; Rodrigues, Matthew A; Lachapelle, Sylvie; Wilkins, Ruth C

    2017-01-01

    Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®(X) (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

  19. Polarized light-scattering profile-advanced characterization of nonspherical particles with scanning flow cytometry.

    Science.gov (United States)

    Strokotov, Dmitry I; Moskalensky, Alexander E; Nekrasov, Vyacheslav M; Maltsev, Valeri P

    2011-07-01

    We instrumentally, theoretically, and experimentally demonstrate a new approach for characterization of nonspherical individual particles from light scattering. Unlike the original optical scheme of the scanning flow cytometer that measures an angle-resolved scattering corresponding in general to S₁₁ element of the light-scattering matrix, the modernized instrument allows us to measure the polarized light-scattering profile of individual particles simultaneously. Theoretically, the polarized profile is expressed by the combination of a few light-scattering matrix elements. This approach supports us with additional independent data to characterize a particle with a complex shape and an internal structure. Applicability of the new method was demonstrated from analysis of polymer bispheres. The bisphere characteristics, sizes, and refractive indices of each sphere composing the bisphere were successfully retrieved from the solution of the inverse light-scattering problem. The solution provides determination of the Eulerian angles, which describe the orientation of the bispheres relative to the direction of the incident laser beam and detecting polarizer of the optical system. Both the ordinary and polarized profiles show a perfect agreement with T-matrix simulation resulting to 50-nm precision for sizing of bispheres. Copyright © 2011 International Society for Advancement of Cytometry.

  20. Assessing microbiological water quality in drinking water distribution systems with disinfectant residual using flow cytometry.

    Science.gov (United States)

    Gillespie, Simon; Lipphaus, Patrick; Green, James; Parsons, Simon; Weir, Paul; Juskowiak, Kes; Jefferson, Bruce; Jarvis, Peter; Nocker, Andreas

    2014-11-15

    Flow cytometry (FCM) as a diagnostic tool for enumeration and characterization of microorganisms is rapidly gaining popularity and is increasingly applied in the water industry. In this study we applied the method to obtain a better understanding of total and intact cell concentrations in three different drinking water distribution systems (one using chlorine and two using chloramines as secondary disinfectants). Chloramine tended to result in lower proportions of intact cells than chlorine over a wider residual range, in agreement with existing knowledge that chloramine suppresses regrowth more efficiently. For chlorinated systems, free chlorine concentrations above 0.5 mg L(-1) were found to be associated with relatively low proportions of intact cells, whereas lower disinfectant levels could result in substantially higher percentages of intact cells. The threshold for chlorinated systems is in good agreement with guidelines from the World Health Organization. The fact that the vast majority of samples failing the regulatory coliform standard also showed elevated proportions of intact cells suggests that this parameter might be useful for evaluating risk of failure. Another interesting parameter for judging the microbiological status of water, the biological regrowth potential, greatly varied among different finished waters providing potential help for investment decisions. For its measurement, a simple method was introduced that can easily be performed by water utilities with FCM capability. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Computationally efficient multidimensional analysis of complex flow cytometry data using second order polynomial histograms.

    Science.gov (United States)

    Zaunders, John; Jing, Junmei; Leipold, Michael; Maecker, Holden; Kelleher, Anthony D; Koch, Inge

    2016-01-01

    Many methods have been described for automated clustering analysis of complex flow cytometry data, but so far the goal to efficiently estimate multivariate densities and their modes for a moderate number of dimensions and potentially millions of data points has not been attained. We have devised a novel approach to describing modes using second order polynomial histogram estimators (SOPHE). The method divides the data into multivariate bins and determines the shape of the data in each bin based on second order polynomials, which is an efficient computation. These calculations yield local maxima and allow joining of adjacent bins to identify clusters. The use of second order polynomials also optimally uses wide bins, such that in most cases each parameter (dimension) need only be divided into 4-8 bins, again reducing computational load. We have validated this method using defined mixtures of up to 17 fluorescent beads in 16 dimensions, correctly identifying all populations in data files of 100,000 beads in analysis, and up to 65 subpopulations of PBMC in 33-dimensional CyTOF data, showing its usefulness in discovery research. SOPHE has the potential to greatly increase efficiency of analysing complex mixtures of cells in higher dimensions.

  2. Recommendations for the development and validation of flow cytometry-based receptor occupancy assays.

    Science.gov (United States)

    Green, Cherie L; Stewart, Jennifer J; Högerkorp, Carl-Magnus; Lackey, Alan; Jones, Nicholas; Liang, Meina; Xu, Yuanxin; Ferbas, John; Moulard, Maxime; Czechowska, Kamila; Mc Closkey, Thomas W; van der Strate, Barry W A; Wilkins, Danice E C; Lanham, David; Wyant, Timothy; Litwin, Virginia

    2016-03-01

    Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.

  3. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

    Science.gov (United States)

    Pasquier, Jennifer; Rioult, Damien; Abu-Kaoud, Nadine; Hoarau-Véchot, Jessica; Marin, Matthieu; Le Foll, Frank

    2015-06-24

    The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  4. Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Jennifer Pasquier

    2015-06-01

    Full Text Available The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp. The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading, we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

  5. Antibiotic susceptibility testing of the Gram-negative bacteria based on flow cytometry

    Directory of Open Access Journals (Sweden)

    Claude Saint-Ruf

    2016-07-01

    Full Text Available Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH, which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3, which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.

  6. Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

    Science.gov (United States)

    Mzabi, Alexandre; Escotte-Binet, Sandie; Le Naour, Richard; Ortis, Naïma; Audonnet, Sandra; Dardé, Marie-Laure; Aubert, Dominique; Villena, Isabelle

    2015-12-01

    The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

  7. Genome Sizes of Nine Insect Species Determined by Flow Cytometry and k-mer Analysis

    Science.gov (United States)

    He, Kang; Lin, Kejian; Wang, Guirong; Li, Fei

    2016-01-01

    The flow cytometry method was used to estimate the genome sizes of nine agriculturally important insects, including two coleopterans, five Hemipterans, and two hymenopterans. Among which, the coleopteran Lissorhoptrus oryzophilus (Kuschel) had the largest genome of 981 Mb. The average genome size was 504 Mb, suggesting that insects have a moderate-size genome. Compared with the insects in other orders, hymenopterans had small genomes, which were averagely about ~200 Mb. We found that the genome sizes of four insect species were different between male and female, showing the organismal complexity of insects. The largest difference occurred in the coconut leaf beetle Brontispa longissima (Gestro). The male coconut leaf beetle had a 111 Mb larger genome than females, which might be due to the chromosome number difference between the sexes. The results indicated that insect invasiveness was not related to genome size. We also determined the genome sizes of the small brown planthopper Laodelphax striatellus (Fallén) and the parasitic wasp Macrocentrus cingulum (Brischke) using k-mer analysis with Illunima Solexa sequencing data. There were slight differences in the results from the two methods. k-mer analysis indicated that the genome size of L. striatellus was 500–700 Mb and that of M. cingulum was ~150 Mb. In all, the genome sizes information presented here should be helpful for designing the genome sequencing strategy when necessary. PMID:27932995

  8. Interaction between carbon nanotubes and mammalian cells: characterization by flow cytometry and application

    Energy Technology Data Exchange (ETDEWEB)

    Cai Dong; Blair, Derek; Dufort, Fay J; Gumina, Maria R; Chiles, Thomas C [Department of Biology, Boston College, Chestnut Hill, MA 02467 (United States); Huang Zhongping; Canahan, D [NanoLab, Incorporated, Newton, MA 02458 (United States); Hong, George [Bioprocess Division, Millipore Corporation, 80 Ashby Road, Bedford, MA 01730 (United States); Wagner, Dean [Naval Health Research Center, Detachment Environmental Health Effects Laboratory, Wright Patterson Air Force Base, OH 45433 (United States); Kempa, K; Ren, Z F [Department of Physics, Boston College, Chestnut Hill, MA 02467 (United States)], E-mail: caid@bc.edu

    2008-08-27

    We show herein that CNT-cell complexes are formed in the presence of a magnetic field. The complexes were analyzed by flow cytometry as a quantitative method for monitoring the physical interactions between CNTs and cells. We observed an increase in side scattering signals, where the amplitude was proportional to the amount of CNTs that are associated with cells. Even after the formation of CNT-cell complexes, cell viability was not significantly decreased. The association between CNTs and cells was strong enough to be used for manipulating the complexes and thereby conducting cell separation with magnetic force. In addition, the CNT-cell complexes were also utilized to facilitate electroporation. We observed a time constant from CNT-cell complexes but not from cells alone, indicating a high level of pore formation in cell membranes. Experimentally, we achieved the expression of enhanced green fluorescence protein by using a low electroporation voltage after the formation of CNT-cell complexes. These results suggest that higher transfection efficiency, lower electroporation voltage, and miniaturized setup dimension of electroporation may be accomplished through the CNT strategy outlined herein.

  9. The clinical utility and prognostic value of multiparameter flow cytometry immunophenotyping in light-chain amyloidosis.

    Science.gov (United States)

    Paiva, Bruno; Vídriales, María-Belén; Pérez, José J; López-Berges, María-Consuelo; García-Sanz, Ramón; Ocio, Enrique M; de Las Heras, Natalia; Cuello, Rebeca; García de Coca, Alfonso; Pardal, Emilia; Alonso, José; Sierra, Magdalena; Bárez, Abelardo; Hernández, José; Suárez, Lissbett; Galende, Josefina; Mateos, María-Victoria; San Miguel, Jesús F

    2011-03-31

    The clinical value of multiparameter flow cytometry (MFC) immunophenotyping in primary or light chain amyloidosis (AL) remains unknown. We studied 44 consecutive bone marrow samples from newly diagnosed patients with amyloidosis; 35 patients with AL and 9 with other forms of amyloidosis. Monoclonal plasma cells (PCs) were identifiable by MFC immunophenotyping in 34 of 35 (97%) patients with AL, whereas it was absent from all but 1 of the 9 (11%) patients with other forms of amyloidosis. Quantification of bone marrow plasma cells (BMPCs) by MFC immunophenotyping was a significant prognostic factor for overall survival (OS) (≤ 1% vs > 1% BMPC cutoff; 2-year OS rates of 90% vs 44%, P = .02). Moreover, detecting persistent normal PCs at diagnosis identifies a subgroup of patients with AL with prolonged OS (> 5% vs ≤ 5% normal PC within all BMPC cutoff, 2-year rates of 88% vs 37%, P = .01). MFC immunophenotyping could be clinically useful for the demonstration of PC clonality in AL and for the prognostication of patients with AL.

  10. DNA and RNA content analysis by flow cytometry in the pathobiologic assessment of bone tumors

    Energy Technology Data Exchange (ETDEWEB)

    El-Naggar, A.K.; Hurr, K.; Tu, Z.N.; Teague, K.; Raymond, K.A.; Ayala, A.G.; Murray, J. [Univ. of Texas, Houston, TX (United States)

    1995-03-01

    Studies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and malignant bone neoplasms. Benign tumors manifested predominantly DNA diploidy and low proliferative activity, whereas the majority of malignant tumors were DNA aneuploid and showed high proliferation rate. No significant difference in the RNA content between different histopathologic categories was found. We observed, however, a distinct and consistently high RNA content pattern in giant cell tumors, aneurysmal bone cysts, and chondroblastomas that may be useful in their differential diagnosis. Analysis of different prognostic factors in malignant tumors indicated that histologic grade and DNA content are significant prognostic factors. Further analysis of malignant tumors showed that a correlation between the proliferative activity and the clinical outcome in the low grade category and between RNA content and patients` survival in osteosarcomas. Our study also showed that preoperative treatment significantly impacted on the extent of the proliferative fraction in malignant tumors. We conclude that DNA/RNA analysis of bone tumor may assist in: (1) the differential diagnosis of certain bone tumors, (2) evaluation of treatment response, and (3) the biological assessment of osteosarcomas. 38 refs., 4 figs., 5 tabs.

  11. Antibacterial Activity of Commercial Dentine Bonding Systems against E. faecalis-Flow Cytometry Study.

    Science.gov (United States)

    Lukomska-Szymanska, Monika; Konieczka, Magdalena; Zarzycka, Beata; Lapinska, Barbara; Grzegorczyk, Janina; Sokolowski, Jerzy

    2017-04-29

    Literature presents inconsistent results on the antibacterial activity of dentine bonding systems (DBS). Antibacterial activity of adhesive systems depends on several factors, including composition and acidity. Flow cytometry is a novel detection method to measure multiple characteristics of a single cell: total cell number, structural (size, shape), and functional parameters (viability, cell cycle). The LIVE/DEAD® BacLightTM bacterial viability assay was used to evaluate an antibacterial activity of DBS by assessing physical membrane disruption of bacteria mediated by DBS. Ten commercial DBSs: four total-etching (TE), four self-etching (SE) and two selective enamel etching (SEE) were tested. Both total-etching DBS ExciTE F and OptiBond Solo Plus showed comparatively low antibacterial activity against E. faecalis. The lowest activity of all tested TE systems showed Te-Econom Bond. Among SE DBS, G-ænial Bond (92.24% dead cells) followed by Clearfil S3 Bond Plus (88.02%) and Panavia F 2.0 ED Primer II (86.67%) showed the highest antibacterial activity against E. faecalis, which was comparable to isopropranol (positive control). In the present study, self-etching DBS exhibited higher antimicrobial activity than tested total-etching adhesives against E. faecalis.

  12. Flow cytometry reticulocyte counting using acridine orange: validation of a new protocol

    Directory of Open Access Journals (Sweden)

    Karina Augusta Viana

    2014-06-01

    Full Text Available Introduction: Currently, the reticulocyte counting is a challenge for clinical laboratories in Brazil, mainly for the ordinary ones, which still use the manual method. This method has some limitations, since it consists of a laborious method, time consuming, with low accuracy. Objectives: This study has developed and evaluated the performance of a New Laboratory Protocol for flow cytometry (FC reticulocytes counting using acridine orange (AO as dye, aiming to standardize a more precise, easy, fast implementation, and low cost protocol. After standardization of the New Protocol (FC/AO, it was compared with the manual method. The results were analyzed according to the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, now known as Clinical and Laboratory Standards Institute (CLSI, to evaluate the interchangeability of methods in linear regression analysis and paired t test, besides other quality control tests. Conclusion: Based on these results concerning to the correlation between the methods and the tests related to quality control, we can admit that FC/AO for reticulocyte counting shows undeniable advantages when compared to the preexisting manual method.

  13. Selection of Aptamers for Mature White Adipocytes by Cell SELEX Using Flow Cytometry

    Science.gov (United States)

    Kim, Eun Young; Kim, Ji Won; Kim, Won Kon; Han, Baek Soo; Park, Sung Goo; Chung, Bong Hyun; Lee, Sang Chul; Bae, Kwang-Hee

    2014-01-01

    Background Adipose tissue, mainly composed of adipocytes, plays an important role in metabolism by regulating energy homeostasis. Obesity is primarily caused by an abundance of adipose tissue. Therefore, specific targeting of adipose tissue is critical during the treatment of obesity, and plays a major role in overcoming it. However, the knowledge of cell-surface markers specific to adipocytes is limited. Methods and Results We applied the CELL SELEX (Systematic Evolution of Ligands by EXponential enrichment) method using flow cytometry to isolate molecular probes for specific recognition of adipocytes. The aptamer library, a mixture of FITC-tagged single-stranded random DNAs, is used as a source for acquiring molecular probes. With the increasing number of selection cycles, there was a steady increase in the fluorescence intensity toward mature adipocytes. Through 12 rounds of SELEX, enriched aptamers showing specific recognition toward mature 3T3-L1 adipocyte cells were isolated. Among these, two aptamers (MA-33 and 91) were able to selectively bind to mature adipocytes with an equilibrium dissociation constant (Kd) in the nanomolar range. These aptamers did not bind to preadipocytes or other cell lines (such as HeLa, HEK-293, or C2C12 cells). Additionally, it was confirmed that MA-33 and 91 can distinguish between mature primary white and primary brown adipocytes. Conclusions These selected aptamers have the potential to be applied as markers for detecting mature white adipocytes and monitoring adipogenesis, and could emerge as an important tool in the treatment of obesity. PMID:24844710

  14. Characterization of Protein Particles in Therapeutic Formulations Using Imaging Flow Cytometry.

    Science.gov (United States)

    Probst, Christine; Zeng, Yuanchun; Zhu, Rong-Rong

    2017-08-01

    Quantitation of particles >10 μm in therapeutic protein formulations is required by pharmacopeia guidelines, and characterization of particles particles; consequently, new methods are needed to measure the sub-10 μm size range. Here, we evaluate imaging flow cytometry (IFC) as a new method for detection of protein aggregates, taking advantage of key enabling attributes including rapid multi-modal high-resolution imaging of individual particles, low sample volume, high sampling efficiency, wide dynamic size and concentration range, and low clog risk. IFC sensitivity was compared with dynamic imaging, a "gold standard" technique for analysis of particles in protein formulations. Both techniques yielded similar results for polystyrene beads ≥2 μm. However, IFC demonstrated greater protein particle detection sensitivity, especially for the sub-10 μm size range. Interestingly, for an aggregated lysozyme sample, IFC detected protein particles using fluorescence images, whereas dynamic imaging failed to detect even large particles >25 μm due to high transparency. The results corroborate implementation of IFC as an advanced technique for protein particle analysis, offering in-depth characterization of particle physical and chemical properties, and enhanced sensitivity for sub-10 μm and transparent particles. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  15. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping.

    Science.gov (United States)

    Freeman, Jennifer L; Adeniyi, Adeola; Banerjee, Ruby; Dallaire, Stephanie; Maguire, Sean F; Chi, Jianxiang; Ng, Bee Ling; Zepeda, Cinthya; Scott, Carol E; Humphray, Sean; Rogers, Jane; Zhou, Yi; Zon, Leonard I; Carter, Nigel P; Yang, Fengtang; Lee, Charles

    2007-06-27

    The zebrafish (Danio rerio) is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG) chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC) clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH) and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.

  16. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

    Directory of Open Access Journals (Sweden)

    Zhou Yi

    2007-06-01

    Full Text Available Abstract Background The zebrafish (Danio rerio is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Results Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. Conclusion The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.

  17. New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

    Science.gov (United States)

    Nava-Castro, Karen; Hernández-Bello, Romel; Muñiz-Hernández, Saé; Escobedo, Galileo; Morales-Montor, Jorge

    2011-01-01

    In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells) is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells. PMID:22187522

  18. Antibacterial Activity of Commercial Dentine Bonding Systems against E. faecalis–Flow Cytometry Study

    Directory of Open Access Journals (Sweden)

    Monika Lukomska-Szymanska

    2017-04-01

    Full Text Available Literature presents inconsistent results on the antibacterial activity of dentine bonding systems (DBS. Antibacterial activity of adhesive systems depends on several factors, including composition and acidity. Flow cytometry is a novel detection method to measure multiple characteristics of a single cell: total cell number, structural (size, shape, and functional parameters (viability, cell cycle. The LIVE/DEAD® BacLightTM bacterial viability assay was used to evaluate an antibacterial activity of DBS by assessing physical membrane disruption of bacteria mediated by DBS. Ten commercial DBSs: four total-etching (TE, four self-etching (SE and two selective enamel etching (SEE were tested. Both total-etching DBS ExciTE F and OptiBond Solo Plus showed comparatively low antibacterial activity against E. faecalis. The lowest activity of all tested TE systems showed Te-Econom Bond. Among SE DBS, G-ænial Bond (92.24% dead cells followed by Clearfil S3 Bond Plus (88.02% and Panavia F 2.0 ED Primer II (86.67% showed the highest antibacterial activity against E. faecalis, which was comparable to isopropranol (positive control. In the present study, self-etching DBS exhibited higher antimicrobial activity than tested total-etching adhesives against E. faecalis.

  19. Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry.

    Science.gov (United States)

    Ukibe, Ken; Katsuragi, Tohoru; Tani, Yoshiki; Takagi, Hiroshi

    2008-09-01

    Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.

  20. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  1. Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

    Science.gov (United States)

    Darzynkiewicz, Zbigniew; Halicka, H. Dorota; Zhao, Hong

    2010-01-01

    This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis. PMID:20687474

  2. A flow-cytometry method for analyzing the composition of membrane rafts.

    Science.gov (United States)

    Morales-García, M Guadalupe; Fournié, Jean-Jacques; Moreno-Altamirano, M Maximina Bertha; Rodríguez-Luna, Gabriela; Flores, Ricardo-Mondragón; Sánchez-García, F Javier

    2008-10-01

    Membrane rafts are involved in a broad variety of biological processes. Their protein composition under growth factor stimulation, anti-inflammatory or proinflammatory microenvironments, or in the course of pathogenic infections still remains to be determined. However, current techniques aimed at the identification of particular proteins on membrane rafts are not devoid of pitfalls. Membrane rafts were obtained by detergent-free based differential centrifugation from Jurkat T cells and J774 macrophages. Membrane rafts were labeled with fluorochrome-labeled antibodies directed against different cell membrane molecules, and with fluorochrome-labeled cholera toxin B that targets GM1 and analyzed by flow cytometry. CD3, CD11a, and GM1 were shown to be differentially expressed on Jurkat T cell-derived membrane rafts, indicating heterogeneity in membrane rafts composition. On the other hand, it was shown in J774 cell-derived membrane rafts that most but not all CD14 is present in the GM1-containing membrane fragments, thus confirming the heterogeneity of membrane rafts composition in other cell lines. The method described here allows the fluorometric assessment of the relative expression of more than one membrane raft component at a time, and at a single vesicle level in a fast and sensitive manner. This method seems to be a suitable approach to evaluate the molecular heterogeneity in membrane rafts composition.

  3. New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

    Directory of Open Access Journals (Sweden)

    Karen Nava-Castro

    2011-01-01

    Full Text Available In parasitology, particularly in helminthes studies, several methods have been used to look for the expression of specific molecules, such as RT-PCR, western blot, 2D-electrophoresis, and microscopy, among others. However, these methods require homogenization of the whole helminth parasite, preventing evaluation of individual cells or specific cell types in a given parasite tissue or organ. Also, the extremely high interaction between helminthes and host cells (particularly immune cells is an important point to be considered. It is really hard to obtain fresh parasites without host cell contamination. Then, it becomes crucial to determine that the analyzed proteins are exclusively from parasitic origin, and not a consequence of host cell contamination. Flow cytometry is a fluorescence-based technique used to evaluate the expression of extra-and intracellular proteins in different type cells, including protozoan parasites. It also allows the isolation and recovery of single-cell populations. Here, we describe a method to isolate and obtain purified helminthes cells.

  4. Quantitative measurement of varicella-zoster virus infection by semiautomated flow cytometry.

    Science.gov (United States)

    Gates, Irina V; Zhang, Yuhua; Shambaugh, Cindy; Bauman, Meredith A; Tan, Charles; Bodmer, Jean-Luc

    2009-04-01

    Varicella-zoster virus (VZV; human herpesvirus 3) is the etiological cause of chickenpox and, upon reactivation from latency, zoster. Currently, vaccines are available to prevent both diseases effectively. A critical requirement for the manufacturing of safe and potent vaccines is the measurement of the biological activity to ensure proper dosing and efficacy, while minimizing potentially harmful secondary effects induced by immunization. In the case of live virus-containing vaccines, such as VZV-containing vaccines, biological activity is determined using an infectivity assay in a susceptible cellular host in vitro. Infectivity measurements generally rely on the enumeration of plaques by visual inspection of an infected cell monolayer. These plaque assays are generally very tedious and labor intensive and have modest throughput and high associated variability. In this study, we have developed a flow cytometry assay to measure the infectivity of the attenuated vaccine strain (vOka/Merck) of VZV in MRC-5 cells with improved throughput. The assay is performed in 96-well tissue culture microtiter plates and is based on the detection and quantification of infected cells expressing VZV glycoproteins on their surfaces. Multiple assay parameters have been investigated, including specificity, limit of detection, limit of quantification, range of linear response, signal-to-noise ratio, and precision. This novel assay appears to be in good concordance with the classical plaque assay results and therefore provides a viable, higher-throughput alternative to the plaque assay.

  5. Platelet Adhesion to Podoplanin Under Flow is Mediated by the Receptor CLEC-2 and Stabilised by Src/Syk-Dependent Platelet Signalling

    Science.gov (United States)

    Pollitt, Alice Y.; Lowe, Kate; Latif, Arusa; Nash, Gerard B.

    2015-01-01

    Summary Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice. PMID:25694214

  6. Blood flow simulation on a role for red blood cells in platelet adhesion

    Science.gov (United States)

    Shimizu, Kazuya; Sugiyama, Kazuyasu; Takagi, Shu

    2016-11-01

    Large-scale blood flow simulations were conducted and a role for red blood cells in platelet adhesion was discussed. The flow conditions and hematocrit values were set to the same as corresponding experiments, and the numerical results were compared with the measurements. Numerical results show the number of platelets adhered on the wall is increased with the increase in hematocrit values. The number of adhered platelets estimated from the simulation was approximately 28 (per 0.01 square millimeter per minute) for the hematocrit value of 20%. These results agree well with the experimental results qualitatively and quantitatively, which proves the validity of the present numerical model including the interaction between fluid and many elastic bodies and the modeling of platelet adhesion. Numerical simulation also reproduces the behavior of red blood cells in the blood flow and their role in platelet adhesion. Red blood cells deform to a flat shape and move towards channel center region. In contrast, platelets are pushed out and have many chances to contact with the wall. As a result, the large number of adhered platelets is observed as hematocrit values becomes high. This result indicates the presence of red blood cells plays a crucial role in platelet adhesion.

  7. Platelet counting with the BD Accuri(TM) C6 flow cytometer.

    Science.gov (United States)

    Masters, Andrew; Harrison, Paul

    2014-01-01

    The Accuri™ C6 is a compact flow cytometer that uses a peristaltic pump with a laminar flow fluidic system and can measure absolute cell counts. In this study we have evaluated this method with the International Reference Method (IRM) simultaneously measured on both the Accuri™ C6 and a reference flow cytometer. After optimisation of sample labelling conditions, final dilutions and flow cytometer settings, a comparison of the absolute fluorescent platelet count with the RBC/platelet ratio on the C6 and the IRM was then performed in 144 patient samples with a full range of platelet counts (range 2-650 × 10(9)/l). The platelet/RBC ratio method determined on the Accuri™ agreed well with the IRM (R(2)=0.99, bias=2.3 (Bland Altman) and R(2)=0.96, bias=1.02 at counts <50 × 10(9)/l). The absolute platelet count also agreed well with the IRM (R(2)=0.97, bias=-0.16 and R(2)=0.91, bias=3.7 at <50 × 10(9)/l). The C6 absolute platelet count and RBC/platelet ratio methods also agreed well (R(2)=0.99, bias=-2.5 and R(2)=0.95, bias=2.71 at counts <50 × 10(9)/l). Reproducibility studies on the C6 gave CVs of <5% for the RB/platelet ratio and <12% for the absolute cell counts. The C6 also demonstrated excellent linearity on diluted samples with both volume and ratio methods (R(2)=0.99). As one might expect, the absolute platelet count is therefore slightly more inaccurate than the RBC/platelet ratio particularly at platelet counts <50 × 10(9)/l as it is likely to be more sensitive to pipetting error. The Accuri™ C6 provides a simple, rapid and reliable method for measuring platelet counts by either the RBC/platelet or direct volume methods. The direct volume method can also be used to determine platelet counts within purified platelet preparations or concentrates in the absence of RBC.

  8. Characterization of typical platelet injector flow configurations. [liquid propellant rocket engines

    Science.gov (United States)

    Hickox, C. E.

    1975-01-01

    A study to investigate the hydraulic atomization characteristics of several novel injector designs for use in liquid propellant rocket engines is presented. The injectors were manufactured from a series of thin stainless steel platelets through which orifices were very accurately formed by a photoetching process. These individual platelets were stacked together and the orifices aligned so as to produce flow passages of prescribed geometry. After alignment, the platelets were bonded into a single, 'platelet injector', unit by a diffusion bonding process. Because of the complex nature of the flow associated with platelet injectors, it was necessary to use experimental techniques, exclusively, throughout the study. Large scale models of the injectors were constructed from aluminum plates and the appropriate fluids were modeled using a glycerol-water solution. Stop-action photographs of test configurations, using spark-shadowgraph or stroboscopic back-lighting, are shown.

  9. Green fiber lasers: An alternative to traditional DPSS green lasers for flow cytometry

    Science.gov (United States)

    Telford, William G.; Babin, Sergey A.; Khorev, Serge V.; Rowe, Stephen H.

    2009-01-01

    Green and yellow diode-pumped solid state (DPSS) lasers (532 and 561 nm) have become common fixtures on flow cytometers, due to their efficient excitation of phycoerythrin (PE) and its tandems, and their ability to excite an expanding array of expressible red fluorescent proteins. Nevertheless, they have some disadvantages. DPSS 532 nm lasers emit very close to the fluorescein bandwidth, necessitating optical modifications to permit detection of fluorescein and GFP. DPSS 561 nm lasers likewise emit very close to the PE detection bandwidth, and also cause unwanted excitation of APC and its tandems, requiring high levels of crossbeam compensation to reduce spectral overlap into the PE tandems. In this paper, we report the development of a new generation of green fiber lasers that can be engineered to emit in the range between 532 and 561 nm. A 550 nm green fiber laser was integrated into both a BD LSR II™ cuvette and FACSVantage DiVa™ jet-in-air cell sorter. This laser wavelength avoided both the fluorescein and PE bandwidths, and provided better excitation of PE and the red fluorescent proteins DsRed and dTomato than a power-matched 532 nm source. Excitation at 550 nm also caused less incidental excitation of APC and its tandems, reducing the need for crossbeam compensation. Excitation in the 550 nm range therefore proved to be a good compromise between 532 and 561 nm sources. Fiber laser technology is therefore providing the flexibility necessary for precisely matching laser wavelengths to our flow cytometry applications. PMID:19777600

  10. Flow cytometry for rapid detection of Salmonella spp. in seed sprouts

    Directory of Open Access Journals (Sweden)

    Bledar Bisha

    2014-12-01

    Full Text Available Seed sprouts (alfalfa, mung bean, radish, etc. have been implicated in several recent national and international outbreaks of salmonellosis. Conditions used for sprouting are also conducive to the growth of Salmonella. As a result, this pathogen can quickly grow to very high cell densities during sprouting without any detectable organoleptic impact. Seed sprouts typically also support heavy growth (~108 CFU g−1 of a heterogeneous microbiota consisting of various bacterial, yeast, and mold species, often dominated by non-pathogenic members of the family Enterobacteriaceae. This heavy background may present challenges to the detection of Salmonella, especially if this pathogen is present in relatively low numbers. We combined DNA-based fluorescence in situ hybridization (FISH with flow cytometry (FCM for the rapid molecular detection of Salmonella enterica ser. Typhimurium in artificially contaminated alfalfa and other seed sprouts. Components of the assay included a set of cooperatively binding probes, a chemical blocking treatment intended to reduce non-specific background, and sample concentration via tangential flow filtration (TFF. We were able to detect S. Typhimurium in sprout wash at levels as low as 103 CFU ml−1 sprout wash (104 CFU g−1 sprouts against high microbial backgrounds (~108 CFU g−1 sprouts. Hybridization times were typically 30 min, with additional washing, but we ultimately found that S. Typhimurium could be readily detected using hybridization times as short as 2 min, without a wash step. These results clearly demonstrate the potential of combined DNA-FISH and FCM for rapid detection of Salmonella in this challenging food matrix and provide industry with a useful tool for compliance with sprout production standards proposed in the Food Safety Modernization Act (FSMA.

  11. Monocyte-Platelet Interaction Induces a Pro-Inflammatory Phenotype in Circulating Monocytes

    OpenAIRE

    2011-01-01

    BACKGROUND: Activated platelets exert a pro-inflammatory action that can be largely ascribed to their ability to interact with leukocytes and modulate their activity. We hypothesized that platelet activation and consequent formation of monocyte-platelet aggregates (MPA) induces a pro-inflammatory phenotype in circulating monocytes. METHODOLOGY/PRINCIPAL FINDINGS: CD62P(+) platelets and MPA were measured, and monocytes characterized, by whole blood flow cytometry in healthy subjects, before an...

  12. Detection of Platelet-Monocyte Aggregates by the ADAM® Image Cytometer

    OpenAIRE

    Jung, Bo Kyeung; Cho, Chi Hyun; Moon, Kyung Chul; sung Hur, Dae; YOON, Jeong-Ah; Yoon, Soo-Young

    2014-01-01

    Background: Inappropriate platelet activation is known to be associated with various thrombotic disorders. Platelet-monocyte aggregates (PMAs), whose formation is mediated by platelet surface P-selectin (CD62P), can be used as a reliable marker to detect platelet activation. Previous studies have generally detected PMAs through flow cytometry-based approaches. Recently, the ADAM® image cytometer (Nanoentek Inc., Seoul, Korea) was developed for image-based cellular analysis. In this study, we ...

  13. Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake

    Directory of Open Access Journals (Sweden)

    Carmen E Contreras

    2004-03-01

    Full Text Available The evaluation of new antimalarial agents using older methods of monitoring sensitivity to antimalarial drugs are laborious and poorly suited to discriminate stage-specific activity. We used flow cytometry to study the effect of established antimalarial compounds, cysteine protease inhibitors, and a quinolone against asexual stages of Plasmodium falciparum. Cultured P. falciparum parasites were treated for 48 h with different drug concentrations and the parasitemia was determined by flow cytometry methods after DNA staining with propidium iodide. P. falciparum erythrocytic life cycle stages were readily distinguished by flow cytometry. Activities of established and new antimalarial compounds measured by flow cytometry were equivalent to results obtained with microscopy and metabolite uptake assays. The antimalarial activity of all compounds was higher against P. falciparum trophozoite stages. Advantages of flow cytometry analysis over traditional assays included higher throughput for data collection, insight into the stage-specificity of antimalarial activity avoiding use of radioactive isotopes.

  14. Quantitative analysis of human platelet adhesions under a small-scale flow device.

    Science.gov (United States)

    Furukawa, Katsuko S; Nakamura, Keigo; Onimura, Yuji; Uchida, Masaki; Ito, Atsuo; Yamane, Takashi; Tamaki, Tamotsu; Ushida, Takashi; Tateishi, Tetsuya

    2010-04-01

    To realize real-time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate-type viscometer, combined with an upright epi-fluorescence microscope. The apparatus allowed real-time evaluation of platelet-material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 microL) under shear flow conditions. To study the dynamic behavior of platelet-material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small-scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large-scale flow system. Therefore, the newly developed cone and plate-type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion.

  15. Whole Blood Activation Results in Altered T Cell and Monocyte Cytokine Production Profiles by Flow Cytometry

    Science.gov (United States)

    Crucian, Brian E.; Sams, Clarence F.

    2001-01-01

    An excellent monitor of the immune balance of peripheral circulating cells is to determine their cytokine production patterns in response to stimuli. Using flow cytometry, a positive identification of cytokine producing cells in a mixed culture may be achieved. Recently, the ability to assess cytokine production following a whole-blood activation culture has been described. In this study, whole blood activation was compared to traditional PBMC activation and the individual cytokine secretion patterns for both T cells, T cell subsets and monocytes was determined by flow cytometry. RESULTS: For T cell cytokine assessment (IFNg/IL-10 and IL-21/L-4) following PMA +ionomycin activation: (1) a Significantly greater percentages of T cells producing IFNgamma and IL-2 were observed following whole-blood culture and (2) altered T cell cytokine production kinetics were observed by varying whole blood culture times. Four-color analysiS was used to allow assessment of cytokine production by specific T cell subsets. It was found that IFNgamma production was significantly elevated in the CD3+/CD8+ T cell population as compared to the CD3+/CD8- population following five hours of whole blood activation. Conversely, IL-2 and IL-10 production were Significantly elevated in the CD3+/CD8- T cell population as compared to the CD3+/CD8+ population. Monocyte cytokine production was assessed in both culture systems following LPS activation for 24 hours. A three-color flow cytometric was used to assess two cytokines (IL-1a/IL-12 and TNFa/IL-10) in conjunction with CD14. Nearly all monocytes were stimulated to produce IL-1a, IL-12 and TNFa. equally well in both culture systems, however monocyte production of IL-10 was significantly elevated in whole blood culture as compared to PBMC culture. IL-12 producing monocytes appeared to be a distinct subpopulation of the IL-1a producing set, whereas IL-10 and TNFa producing monocytes were largely mutually exclusive. IL-10 and TNFa producing

  16. Modelling of platelet-fibrin clot formation in flow with a DPD-PDE method.

    Science.gov (United States)

    Tosenberger, A; Ataullakhanov, F; Bessonov, N; Panteleev, M; Tokarev, A; Volpert, V

    2016-02-01

    The paper is devoted to mathematical modelling of clot growth in blood flow. Great complexity of the hemostatic system dictates the need of usage of the mathematical models to understand its functioning in the normal and especially in pathological situations. In this work we investigate the interaction of blood flow, platelet aggregation and plasma coagulation. We develop a hybrid DPD-PDE model where dissipative particle dynamics (DPD) is used to model plasma flow and platelets, while the regulatory network of plasma coagulation is described by a system of partial differential equations. Modelling results confirm the potency of the scenario of clot growth where at the first stage of clot formation platelets form an aggregate due to weak inter-platelet connections and then due to their activation. This enables the formation of the fibrin net in the centre of the platelet aggregate where the flow velocity is significantly reduced. The fibrin net reinforces the clot and allows its further growth. When the clot becomes sufficiently large, it stops growing due to the narrowed vessel and the increase of flow shear rate at the surface of the clot. Its outer part is detached by the flow revealing the inner part covered by fibrin. This fibrin cap does not allow new platelets to attach at the high shear rate, and the clot stops growing. Dependence of the final clot size on wall shear rate and on other parameters is studied.

  17. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Galanzha, Ekaterina I. [Phillips Classic Laser and Nanomedicine Laboratories, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Zharov, Vladimir P., E-mail: zharovvladimirp@uams.edu [Phillips Classic Laser and Nanomedicine Laboratories, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States); Arkansas Nanomedicine Center, University of Arkansas for Medical Sciences, 4301 West Markham Street, Little Rock, AR 72205 (United States)

    2013-12-10

    Despite progress in detecting circulating tumor cells (CTCs), existing assays still have low sensitivity (1–10 CTC/mL) due to the small volume of blood samples (5–10 mL). Consequently, they can miss up to 10{sup 3}–10{sup 4} CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 10{sup 2}–10{sup 3} times) by the examination of the entire blood volume in vivo (5 L in adults). We focus on in vivo photoacoustic (PA) flow cytometry (PAFC) of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL) and throughput (up to 10 mL/min) than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-resolution PAFC beyond the diffraction and spectral limits.

  18. Circulating Tumor Cell Detection and Capture by Photoacoustic Flow Cytometry in Vivo and ex Vivo

    Directory of Open Access Journals (Sweden)

    Ekaterina I. Galanzha

    2013-12-01

    Full Text Available Despite progress in detecting circulating tumor cells (CTCs, existing assays still have low sensitivity (1–10 CTC/mL due to the small volume of blood samples (5–10 mL. Consequently, they can miss up to 103–104 CTCs, resulting in the development of barely treatable metastasis. Here we analyze a new concept of in vivo CTC detection with enhanced sensitivity (up to 102–103 times by the examination of the entire blood volume in vivo (5 L in adults. We focus on in vivo photoacoustic (PA flow cytometry (PAFC of CTCs using label-free or targeted detection, photoswitchable nanoparticles with ultrasharp PA resonances, magnetic trapping with fiber-magnetic-PA probes, optical clearance, real-time spectral identification, nonlinear signal amplification, and the integration with PAFC in vitro. We demonstrate PAFC’s capability to detect rare leukemia, squamous carcinoma, melanoma, and bulk and stem breast CTCs and its clusters in preclinical animal models in blood, lymph, bone, and cerebrospinal fluid, as well as the release of CTCs from primary tumors triggered by palpation, biopsy or surgery, increasing the risk of metastasis. CTC lifetime as a balance between intravasation and extravasation rates was in the range of 0.5–4 h depending on a CTC metastatic potential. We introduced theranostics of CTCs as an integration of nanobubble-enhanced PA diagnosis, photothermal therapy, and feedback through CTC counting. In vivo data were verified with in vitro PAFC demonstrating a higher sensitivity (1 CTC/40 mL and throughput (up to 10 mL/min than conventional assays. Further developments include detection of circulating cancer-associated microparticles, and super-rsesolution PAFC beyond the diffraction and spectral limits.

  19. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study

    Science.gov (United States)

    Ogata, Kiyoyuki; Della Porta, Matteo G.; Malcovati, Luca; Picone, Cristina; Yokose, Norio; Matsuda, Akira; Yamashita, Taishi; Tamura, Hideto; Tsukada, Junichi; Dan, Kazuo

    2009-01-01

    Background The diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. Design and Methods We designed a flow cytometry protocol applicable in many laboratories and verified its diagnostic utility in patients without those diagnostic markers. The cardinal parameters, analyzable from one cell aliquot, were myeloblasts (%), B-cell progenitors (%), myeloblast CD45 expression, and channel number of side scatter where the maximum number of granulocytes occurs. The adjunctive parameters were CD11b, CD15, and CD56 expression (%) on myeloblasts. Marrow samples from 106 control patients with cytopenia and 134 low-grade myelodysplastic syndromes patients, including 81 lacking both ringed sideroblasts and cytogenetic aberrations, were prospectively analyzed in Japan and Italy. Results Data outside the predetermined reference range in 2 or more parameters (multiple abnormalities) were common in myelodysplastic syndromes patients. In those lacking ringed sideroblasts and cytogenetic aberrations, multiple abnormalities were observed in 8/26 Japanese (30.8%) and 37/55 Italians (67.3%) when the cardinal parameters alone were considered, and in 17/26 Japanese (65.4%) and 42/47 Italians (89.4%) when all parameters were taken into account. Multiple abnormalities were rare in controls. When data from all parameters were used, the diagnostic sensitivities were 65% and 89%, specificities were 98% and 90%, and likelihood ratios were 28.1 and 8.5 for the Japanese and Italian cohorts, respectively. Conclusions This protocol can be used in the diagnostic work-up of low-grade myelodysplastic syndromes patients who lack specific diagnostic markers, although further improvement in diagnostic power is desirable. PMID:19546439

  20. Coconut genome size determined by flow cytometry: Tall versus Dwarf types.

    Science.gov (United States)

    Freitas Neto, M; Pereira, T N S; Geronimo, I G C; Azevedo, A O N; Ramos, S R R; Pereira, M G

    2016-02-11

    Coconuts (Cocos nucifera L.) are tropical palm trees that are classified into Tall and Dwarf types based on height, and both types are diploid (2n = 2x = 32 chromosomes). The reproduction mode is autogamous for Dwarf types and allogamous for Tall types. One hypothesis for the origin of the Dwarf coconut suggests that it is a Tall variant that resulted from either mutation or inbreeding, and differences in genome size between the two types would support this hypothesis. In this study, we estimated the genome sizes of 14 coconut accessions (eight Tall and six Dwarf types) using flow cytometry. Nuclei were extracted from leaf discs and stained with propidium iodide, and Pisum sativum (2C = 9.07 pg DNA) was used as an internal standard. Histograms with good resolution and low coefficients of variation (2.5 to 3.2%) were obtained. The 2C DNA content ranged from 5.72 to 5.48 pg for Tall accessions and from 5.58 to 5.52 pg for Dwarf accessions. The mean genome sizes for Tall and Dwarf specimens were 5.59 and 5.55 pg, respectively. Among all accessions, Rennel Island Tall had the highest mean DNA content (5.72 pg), whereas West African Tall had the lowest (5.48 pg). The mean coconut genome size (2C = 5.57 pg, corresponding to 2723.73 Mbp/haploid set) was classified as small. Only small differences in genome size existed among the coconut accessions, suggesting that the Dwarf type did not evolve from the Tall type.

  1. Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer.

    Directory of Open Access Journals (Sweden)

    Cristiane S Ferreira-Facio

    Full Text Available Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples, with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 35, with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56(hi/GD2(+/CD81(hi, primitive neuroectodermal tumors (CD271(hi/CD99(+, Wilms tumors (>1 cell population, rhabdomyosarcoma (nuMYOD1(+/numyogenin(+, carcinomas (CD45(-/EpCAM(+, germ cell tumors (CD56(+/CD45(-/NG2(+/CD10(+ and eventually also hemangiopericytomas (CD45(-/CD34(+. In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

  2. Development of a novel cell sorting method that samples population diversity in flow cytometry.

    Science.gov (United States)

    Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

    2015-11-01

    Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance.

  3. Binding of semenogelin I to intact human spermatozoa studied by flow cytometry and surface plasmon resonance.

    Science.gov (United States)

    Jonsson, Magnus; Frohm, Birgitta; Malm, Johan

    2010-01-01

    Approximately 1 in 10 couples is infertile. No definite cause can be found in about 25% of those cases. Studies have indicated that seminal vesicle secretion functions as an optimizer of fertilization. The Zn(2+) binding protein semenogelin I (SgI) represents a major fraction of the proteins present in seminal vesicle fluid, and it serves as a structural component of the coagulum that is formed after ejaculation. Cleavage of SgI by prostate-specific antigen results in liquefaction of the coagulum. Fragmented SgI has antibacterial effects and inhibits spermatozoa mobility. SgI has also been found complexed to eppin on spermatozoa, and this complex has been suggested to be of importance for fertility. Here, we used flow cytometry and surface plasmon resonance to study SgI regarding its association with spermatozoa and the interaction dependency on Zn(2+). The concentration of Zn(2+) in seminal plasma is approximately 100 times higher than in blood plasma, and the metal ion is known to change the structure of SgI. We found that SgI binds to spermatozoa in a concentration-dependent and saturable manner. In solution, SgI bound to spermatozoa in a non-Zn(2+)-dependent way, whereas immobilized SgI interacts with spermatozoa only in the presence of Zn(2+). It indicates that SgI must exhibit a specific structure or free flexibility to be able to interact with that ligand. Our results indicate that the association of SgI to spermatozoa is conformation dependent and specific. These findings could constitute a basis for the development of a male contraceptive.

  4. Evaluation of a Prototype Flow Cytometry Test for Serodiagnosis of Canine Visceral Leishmaniasis

    Science.gov (United States)

    Ker, Henrique Gama; Coura-Vital, Wendel; Aguiar-Soares, Rodrigo Dian de Oliveira; Roatt, Bruno Mendes; das Dores Moreira, Nádia; Carneiro, Cláudia Martins; Machado, Evandro Marques de Menezes; Teixeira-Carvalho, Andréa; Martins-Filho, Olindo Assis; Giunchetti, Rodolfo Cordeiro; Araújo, Márcio Sobreira Silva; Coelho, Eduardo Antonio Ferraz; da Silveira-Lemos, Denise

    2013-01-01

    Diagnosing canine visceral leishmaniasis (CVL) is a critical challenge since conventional immunoserological tests still present some deficiencies. The current study evaluated a prototype flow cytometry serology test, using antigens and fluorescent antibodies that had been stored for 1 year at 4°C, on a broad range of serum samples. Noninfected control dogs and Leishmania infantum-infected dogs were tested, and the prototype test showed excellent performance in differentiating these groups with high sensitivity, specificity, positive and negative predictive values, and accuracy (100% in all analyses). When the CVL group was evaluated according to the dogs' clinical status, the prototype test showed outstanding accuracy in all groups with positive serology (asymptomatic II, oligosymptomatic, and symptomatic). However, in dogs which had positive results by PCR-restriction fragment length polymorphism (RFLP) but negative results by conventional serology (asymptomatic I), serological reactivity was not observed. Additionally, sera from 40 dogs immunized with different vaccines (Leishmune, Leish-Tec, or LBSap) did not present serological reactivity in the prototype test. Eighty-eight dogs infected with other pathogens (Trypanosoma cruzi, Leishmania braziliensis, Ehrlichia canis, and Babesia canis) were used to determine cross-reactivity and specificity, and the prototype test performed well, particularly in dogs infected with B. canis and E. canis (100% and 93.3% specificities, respectively). In conclusion, our data reinforce the potential of the prototype test for use as a commercial kit and highlight its outstanding performance even after storage for 1 year at 4°C. Moreover, the prototype test efficiently provided accurate CVL serodiagnosis with an absence of false-positive results in vaccinated dogs and minor cross-reactivity against other canine pathogens. PMID:24108778

  5. Flow cytometry for the analysis of α-dystroglycan glycosylation in fibroblasts from patients with dystroglycanopathies.

    Directory of Open Access Journals (Sweden)

    Elizabeth Stevens

    Full Text Available α-dystroglycan (α-DG is a peripheral membrane protein that is an integral component of the dystrophin-glycoprotein complex. In an inherited subset of muscular dystrophies known as dystroglycanopathies, α-DG has reduced glycosylation which results in lower affinity binding to several extracellular matrix proteins including laminins. The glycosylation status of α-DG is normally assessed by the binding of the α-DG antibody IIH6 to a specific glycan epitope on α-DG involved in laminin binding. Immunocytochemistry and immunoblotting are two of the most widely used methods to detect the amount of α-DG glycosylation in muscle. While the interpretation of the presence or absence of the epitope on muscle using these techniques is straightforward, the assessment of a mild defect can be challenging. In this study, flow cytometry was used to compare the amount of IIH6-reactive glycans in fibroblasts from dystroglycanopathy patients with defects in genes known to cause α-DG hypoglycosylation to the amount in fibroblasts from healthy and pathological control subjects. A total of twenty one dystroglycanopathy patient fibroblasts were assessed, as well as fibroblasts from three healthy controls and seven pathological controls. Control fibroblasts have clearly detectable amounts of IIH6-reactive glycans, and there is a significant difference in the amount of this glycosylation, as measured by the mean fluorescence intensity of an antibody recognising the epitope and the percentage of cells positive for the epitope, between these controls and dystroglycanopathy patient fibroblasts (p<0.0001 for both. Our results indicate that the amount of α-DG glycosylation in patient fibroblasts is comparable to that in patient skeletal muscle. This method could complement existing immunohistochemical assays in skeletal muscle as it is quantitative and simple to perform, and could be used when a muscle biopsy is not available. This test could also be used to assess the

  6. Identification and characterization of neutrophil extracellular trap shapes in flow cytometry

    Science.gov (United States)

    Ginley, Brandon; Emmons, Tiffany; Sasankan, Prabhu; Urban, Constantin; Segal, Brahm H.; Sarder, Pinaki

    2017-03-01

    Neutrophil extracellular trap (NET) formation is an alternate immunologic weapon used mainly by neutrophils. Chromatin backbones fused with proteins derived from granules are shot like projectiles onto foreign invaders. It is thought that this mechanism is highly anti-microbial, aids in preventing bacterial dissemination, is used to break down structures several sizes larger than neutrophils themselves, and may have several more uses yet unknown. NETs have been implied to be involved in a wide array of systemic host immune defenses, including sepsis, autoimmune diseases, and cancer. Existing methods used to visually quantify NETotic versus non-NETotic shapes are extremely time-consuming and subject to user bias. These limitations are obstacles to developing NETs as prognostic biomarkers and therapeutic targets. We propose an automated pipeline for quantitatively detecting neutrophil and NET shapes captured using a flow cytometry-imaging system. Our method uses contrast limited adaptive histogram equalization to improve signal intensity in dimly illuminated NETs. From the contrast improved image, fixed value thresholding is applied to convert the image to binary. Feature extraction is performed on the resulting binary image, by calculating region properties of the resulting foreground structures. Classification of the resulting features is performed using Support Vector Machine. Our method classifies NETs from neutrophils without traps at 0.97/0.96 sensitivity/specificity on n = 387 images, and is 1500X faster than manual classification, per sample. Our method can be extended to rapidly analyze whole-slide immunofluorescence tissue images for NET classification, and has potential to streamline the quantification of NETs for patients with diseases associated with cancer and autoimmunity.

  7. UVB-Radiation-Induced Apoptosis in Jurkat Cells: A Coordinated Fourier Transform Infrared Spectroscopy-Flow Cytometry Study

    CERN Document Server

    Pozzi, Deleana; Gaudenzi, Silvia; Di Giambattista, Lucia; Silvestri, Ida; Morrone, Stefania; Castellano, Agostina Congiu

    2010-01-01

    We studied the induction of apoptosis in Jurkat cells by UVB radiation (wavelength 290-320 nm) at a dose of 310 mJ/cm^2. We combined Fourier transform infrared (FTIR) spectroscopy with flow cytometry to determine whether the combination of both techniques could provide new and improved information about cell modifications. To do this, we looked for correspondences and correlations between spectroscopy and flow cytometry data and found three highly probable spectroscopic markers of apoptosis. The behavior of the wave number shift of both the Amide I beta-sheet component and the area of the 1083 cm^-1 band reproduced, with a high correlation, the behavior of the early apoptotic cell population, while the behavior of the Amide I area showed a high correlation with the early plus late apoptotic cell population.

  8. A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

    Directory of Open Access Journals (Sweden)

    L.P.S. Alves

    Full Text Available The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i cell permeabilization, ii Nile red staining, and iii analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99 compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

  9. Quantitative assessment of toxic and nontoxic Microcystis colonies in natural environments using fluorescence in situ hybridization and flow cytometry

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Toxic cyanobacterial blooms constitute a threat to human safety because Microcystis sp. releases microcystins during growth, and particularly during cell death. Therefore, analysis of toxic and nontoxic Microcystis in natural communities is required in order to assess and predict bloom dynamics and toxin production by these organisms. In this study, an analysis combining fluorescence in situ hybridization (FISH) with flow cytometry (FCM) was used to discriminate between toxic and nontoxic Microcystis and also to quantify the percentage of toxic Microcystis present in blooms. The results demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of Microcystis toxin production and for providing an early warning for toxic Microcystis blooms.

  10. Flow Cytometry-Assisted Cloning of Specific Sequence Motifs from Complex 16S rRNA Gene Libraries

    DEFF Research Database (Denmark)

    Nielsen, Jeppe Lund; Schramm, Andreas; Bernhard, Anne E.

    2004-01-01

      FLOW CYTOMETRY-ASSISTED CLONING OF SPECIFIC SEQUENCE MOTIFS FROM COMPLEX 16S RRNA GENE LIBRARIES Jeppe L. Nielsen,1 Andreas Schramm,1,2 Anne E. Bernhard,1 Gerrit J. van den Engh,3 and David A. Stahl1* Department of Civil and Environmental Engineering, University of Washington,1 and Institute...... for Systems Biology,3 Seattle, Washington, and Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany2 A flow cytometry method was developed for rapid screening and recovery of cloned DNA containing common sequence motifs. This approach, termed fluorescence-activated cell sorting......-assisted cloning, was used to recover sequences affiliated with a unique lineage within the Bacteroidetes not abundant in a clone library of environmental 16S rRNA genes.  ...

  11. Lymphocyte and monocyte flow cytometry immunophenotyping as a diagnostic tool in uncharacteristic inflammatory disorders

    Directory of Open Access Journals (Sweden)

    Grip Olof

    2010-07-01

    Full Text Available Abstract Background Patients with uncharacteristic inflammatory symptoms such as long-standing fatigue or pain, or a prolonged fever, constitute a diagnostic and therapeutic challenge. The aim of the present study was to determine if an extended immunophenotyping of lymphocytes and monocytes including activation markers can define disease-specific patterns, and thus provide valuable diagnostic information for these patients. Methods Whole blood from patients with gram-negative bacteraemia, neuroborreliosis, tuberculosis, acute mononucleosis, influenza or a mixed connective tissue disorders, as diagnosed by routine culture and serology techniques was analysed for lymphocyte and monocyte cell surface markers using a no-wash, no-lyse protocol for multi-colour flow cytometry method. The immunophenotyping included the activation markers HLA-DR and CD40. Plasma levels of soluble TNF alpha receptors were analysed by ELISA. Results An informative pattern was obtained by combining two of the analysed parameters: (i, the fractions of HLA-DR-expressing CD4+ T cells and CD8+ T cells, respectively, and (ii, the level of CD40 on CD14+ CD16- monocytes. Patients infected with gram-negative bacteria or EBV showed a marked increase in monocyte CD40, while this effect was less pronounced for tuberculosis, borrelia and influenza. The bacterial agents could be distinguished from the viral agents by the T cell result; CD4+ T cells reacting in bacterial infection, and the CD8+ T cells dominating for the viruses. Patients with mixed connective tissue disorders also showed increased activation, but with similar engagement of CD4+ and CD8+ T cells. Analysis of soluble TNF alpha receptors was less informative due to a large inter-individual variation. Conclusion Immunophenotyping including the combination of the fractions of HLA-DR expressing T cell subpopulations with the level of CD40 on monocytes produces an informative pattern, differentiating between infections of

  12. Blood group antigen studies using CdTe quantum dots and flow cytometry

    Directory of Open Access Journals (Sweden)

    Cabral Filho PE

    2015-07-01

    Full Text Available Paulo E Cabral Filho,1 Maria IA Pereira,1 Heloise P Fernandes,2 Andre A de Thomaz,3 Carlos L Cesar,3 Beate S Santos,4 Maria L Barjas-Castro,2 Adriana Fontes1 1Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco, Recife, Pernambuco, 2Centro de Hematologia e Hemoterapia, Universidade Estadual de Campinas, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, 3Departamento de Eletrônica Quântica, Instituto de Física Gleb Wataghin, Universidade Estadual de Campinas, Campinas, São Paulo, 4Departamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, PE, Brazil Abstract: New methods of analysis involving semiconductor nanocrystals (quantum dots [QDs] as fluorescent probes have been highlighted in life science. QDs present some advantages when compared to organic dyes, such as size-tunable emission spectra, broad absorption bands, and principally exceptional resistance to photobleaching. Methods applying QDs can be simple, not laborious, and can present high sensibility, allowing biomolecule identification and quantification with high specificity. In this context, the aim of this work was to apply dual-color CdTe QDs to quantify red blood cell (RBC antigen expression on cell surface by flow cytometric analysis. QDs were conjugated to anti-A or anti-B monoclonal antibodies, as well as to the anti-H (Ulex europaeus I lectin, to investigate RBCs of A1, B, A1B, O, A2, and Aweak donors. Bioconjugates were capable of distinguishing the different expressions of RBC antigens, both by labeling efficiency and by flow cytometry histogram profile. Furthermore, results showed that RBCs from Aweak donors present fewer amounts of A antigens and higher amounts of H, when compared to A1 RBCs. In the A group, the amount of A antigens decreased as A1 > A3 > AX = Ael, while H antigens were AX = Ael > A1. Bioconjugates presented stability and remained active for at least 6 months. In conclusion

  13. [Function of flow cytometry on the dosage of antibodies against double stranded DNA in systemic lupus erythematosus].

    Science.gov (United States)

    Ferrero, Paola V; Drenkard, Cristina; Collino, César; Cabral, María José; Gamron, Susana; Barberis, Gloria; Onetti, Carlos M; Menso de Ezcurra, Emilia M

    2005-01-01

    Among the diverse number of antibodies observed in systemic lupus erythematosus, antibodies against double stranded DNA (anti-dsDNA) represent important serologic markers for the disease diagnosis and the follow-up of the disease activity. To evaluate the role of a new quantitative methodology to detect antibodies against double stranded DNA in systemic lupus erythematosus and its association with the disease activity. The performance of the indirect immunofluorescence flow cytometry with Crithidia luciliae as substrate was compared with the Crithidia luciliae indirect immunofluorescence assay and the ELISA technique in order to detect antibodies against double stranded DNA in 54 sera from 47 patient with systemic lupus erythematosus and 100 sera from normal controls. The new method showed a sensitivity of 78% and a specificity of 81% when the Crithidia luciliae indirect immunofluorescence assay was the gold standard. Compared with the ELISA technique, the flow cytometry showed a sensitivity of 78% and a specificity of 86%. No correlation was found among antibodies against double stranded DNA values detected with flow cytometry and the MEX-SLEDAI activity scores. However, the flow cytometry showed a sensitivity of 70% and a specificity of 42% to distinguish patients with systemic lupus erythematosus with and without activity (MEX-SLEDAI score > or = 5). The Rho intra-observer coefficient was 0.61 (p < 0.0001). In spite of the fact that this new method might represent an interesting advance for antibodies against double stranded DNA quantitative testing, a clear superiority does not emerge when it was compared with more traditional assays. Difficulties related with its reproducibility might represent a limitation in the routine use of this new method.

  14. Detection and analysis of two serotypes of ammonia-oxidizing bacteria in sewage plants by flow cytometry.

    OpenAIRE

    Völsch, A; Nader, W F; Geiss, H. K.; Nebe, G; Birr, C

    1990-01-01

    Two different serotypes of the genus Nitrosomonas were isolated from samples of the sewage plant Heidelberg. These nitrifiers were enumerated in activated sludge of various other sewage plants after immunofluorescent labeling and staining with propidium iodide by flow cytometry. The concentrations of these serotypes of Nitrosomonas spp. were in the range of 0.1 to 2%. Also, a test for the determination of the activity of ammonia-oxidizing bacteria was developed. Nitrite-oxidizing bacteria wer...

  15. Application of a Short Intracellular pH Method to Flow Cytometry for Determining Saccharomyces cerevisiae Vitality ▿

    OpenAIRE

    Weigert, Claudia; Steffler, Fabian; Kurz, Tomas; Shellhammer, Thomas H.; Methner, Frank-Jürgen

    2009-01-01

    The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and station...

  16. Multiparametric Flow Cytometry and Cell Sorting for the Assessment of Viable, Injured, and Dead Bifidobacterium Cells during Bile Salt Stress

    OpenAIRE

    2002-01-01

    Using a flow cytometry-based approach, we assessed the viability of Bifidobacterium lactis DSM 10140 and Bifidobacterium adolescentis DSM 20083 during exposure to bile salt stress. Carboxyfluorescein diacetate (cFDA), propidium iodide (PI), and oxonol [DiBAC4(3)] were used to monitor esterase activity, membrane integrity, and membrane potential, respectively, as indicators of bacterial viability. Single staining with these probes rapidly and noticeably reflected the behavior of the two strain...

  17. Proposal for the standardization of flow cytometry protocols to detect minimal residual disease in acute lymphoblastic leukemia

    Science.gov (United States)

    Ikoma, Maura Rosane Valério; Beltrame, Miriam Perlingeiro; Ferreira, Silvia Inês Alejandra Cordoba Pires; Souto, Elizabeth Xisto; Malvezzi, Mariester; Yamamoto, Mihoko

    2015-01-01

    Minimal residual disease is the most powerful predictor of outcome in acute leukemia and is useful in therapeutic stratification for acute lymphoblastic leukemia protocols. Nowadays, the most reliable methods for studying minimal residual disease in acute lymphoblastic leukemia are multiparametric flow cytometry and polymerase chain reaction. Both provide similar results at a minimal residual disease level of 0.01% of normal cells, that is, detection of one leukemic cell in up to 10,000 normal nucleated cells. Currently, therapeutic protocols establish the minimal residual disease threshold value at the most informative time points according to the appropriate methodology employed. The expertise of the laboratory in a cancer center or a cooperative group could be the most important factor in determining which method should be used. In Brazil, multiparametric flow cytometry laboratories are available in most leukemia treatment centers, but multiparametric flow cytometry processes must be standardized for minimal residual disease investigations in order to offer reliable and reproducible results that ensure quality in the clinical application of the method. The Minimal Residual Disease Working Group of the Brazilian Society of Bone Marrow Transplantation (SBTMO) was created with that aim. This paper presents recommendations for the detection of minimal residual disease in acute lymphoblastic leukemia based on the literature and expertise of the laboratories who participated in this consensus, including pre-analytical and analytical methods. This paper also recommends that both multiparametric flow cytometry and polymerase chain reaction are complementary methods, and so more laboratories with expertise in immunoglobulin/T cell receptor (Ig/TCR) gene assays are necessary in Brazil. PMID:26670404

  18. Multicolor flow cytometry analysis of blood cell subsets in patients given total body irradiation before bone marrow transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Clave, E.; Socie, G.; Carosella, E. [Hopital-Saint Louis, Paris (France)] [and others

    1995-11-01

    Bone marrow transplantation has often been closely linked with accidental or intentional therapeutical irradiation. In both situations, study of the radiosensitivity of human blood cell subsets is of interest. Using one-color flow cytometry analysis of B lymphocytes, T cell subsets, and natural killer cells, we previously reported that lymphocyte subsets exhibit equal radiosensitivity. Taking advantage of recent developments in the knowledge of leukocyte differentiation antigens and flow cytometry technology we undertook a study of blood cell subsets to search for rare populations exhibiting different radiosensitivity. Thirty patients, who were delivered a 12 Gy fractionated total body irradiation as part of their conditioning regimen before transplantation for malignant disorders, were studied using multicolor flow cytometry. T and B lymphocytes showed a sharp, radiation-induced decrease, with the B lymphocytes (cluster of differentiation (CD) 19+) being the most sensitive. When analyzed by multicolor flow cytometry all major lymphocyte subsets appeared equally sensitive to the in vivo irradiation. Therefore, all major lymphocyte subsets sharing the helper phenotype (naive or memory) and the cytotoxic phenotype appeared equally sensitive to in vivo whole body irradiation. In parallel, the CD34+ cell subset remained basically unchanged after whole body irradiation. Finally, the CD3{minus}, 56+, 16+ natural killer cell subset was relatively radioresistant (91 and 74% of its initial value, after 2 and 4 Gy, respectively) as compared to other lymphocyte subsets. Our study provides evidence that T and B cell subsets seem to be highly radiosensitive in vivo. The CD34+ progenitor/stem cells and NK cells seem to be more radioresistant. This latter result might provide clues to the understanding of the pathophysiogeny of radiation-induced aplasia and of the engrafment/rejection process following bone marrow transplantation. 20 refs., 3 figs., 1 tab.

  19. Flow cytometry is of limited utility in the early identification of "double-hit" B-cell lymphomas.

    Science.gov (United States)

    Platt, Mia Y; DeLelys, Michelle E; Preffer, Frederic I; Sohani, Aliyah R

    2013-05-01

    B-cell lymphomas with concurrent translocations of MYC and BCL2 or BCL6, also known as "double-hit" lymphomas (DHL), are rare malignancies characterized by aggressive clinical behavior and poor prognosis. Previous reports suggest that decreased CD20 and/or CD19 expression by flow cytometry is relatively common in DHL and may help to identify cases requiring additional cytogenetic analysis. We conducted a retrospective analysis of 26 cases of DHL, and compared their flow cytometric characteristics to cases of Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Cases were analyzed by four-color flow cytometry, and bivariate dot-plots were reviewed for light scatter characteristics, CD19, CD20, CD45, and surface light chain. Relatively few DHL cases showed dim expression of CD19 or CD20, and statistically significant differences were found only in the frequency of dim CD19 expression between DHL and BL or DLBCL. Although concomitant dim CD19 and CD20 expression was exclusive to DHL, it was present in only a minority of cases. We conclude that although a subset of DHL expresses aberrant levels of CD19 and/or CD20 by flow cytometry, these findings are of limited utility in identifying cases requiring cytogenetic analysis due to their low frequency. Until more sensitive pathologic parameters can be identified and validated, the decision to perform cytogenetic analysis should rest on a combination of clinical, morphologic, and immunophenotypic features suggestive of high-grade, aggressive disease. Copyright © 2013 International Clinical Cytometry Society.

  20. Detection of IgG Anti-Leishmania Antigen by Flow Cytometry as a Diagnostic Test for Cutaneous Leishmaniasis

    Science.gov (United States)

    Schriefer, Albert; Magalhães, Andréa; Meyer, Roberto; Glesby, Marshall J.; Carvalho, Edgar M.; Carvalho, Lucas P.

    2016-01-01

    Diagnosis of cutaneous leishmaniasis (CL) relies on clinical presentation, parasite isolation, histopathologic evaluation and positive Montenegro skin test. However, the low amounts of parasites in the lesion of these individuals make parasite isolation and histopatologic diagnosis unreliable, often leading to false-negative results. Also, 15% of people living in endemic areas have sub-clinical infection characterized by positive Montenegro skin test, which may contribute to misdiagnosis. Although the main Leishmania killing mechanism is through cell-mediated immune response, antibodies against Leishmania antigens are found in infected individuals. Here our goal was to develop a new serological technique using polystyrene microspheres sensitized with soluble Leishmania antigens as a tool for the detection of IgG in serum from CL patients by flow cytometry. To validate the assay we carried out a comparative test (ELISA) commonly used as a diagnostic test for parasitic diseases. To determine cross-reactivity we used serum from patients with Chagas disease, caused by a trypanosome that has several proteins with high homology to those of the Leishmania genus. We observed that the flow cytometry technique was more sensitive than the ELISA, but, less specific. Our results show that the flow cytometry serologic test can be used to confirm CL cases in L. braziliensis transmission areas, however, presence of Chagas disease has to be ruled out in these individuals. PMID:27622535

  1. Survey of Anti-Bacterial Effect of Plant Extracts (Fennel-Dill-Caraway-Cinnamon by Flow Cytometry and Disk Diffusion

    Directory of Open Access Journals (Sweden)

    P. Ranjbarian

    2004-10-01

    Full Text Available H.pylori has been discovered as an etiologic agent for peptic ulcer and gastric cancer. It is well known that eradicating H.pylori is an essential step in curing ulcer disease. Many regimens are currently available but none of them can achieve 100% eradication rate . In this research, anti-bacterial effect of extracts of:fennel, dill, caraway, cinnamon and antibiotics of: ciprofloxacin, Tetracyclin and amoxycillin were investigated against H.pylori by disk diffusion method and flow cytometry. In this study we used culture and rapid urease Test , catalase, oxidase and also staining to recognize H.pylori in 30 biopsies that has been taken from patients . 14 cases (46.66% were positive for H.pylori infection. Disk diffusion method was used to detremine the sensitivity of H.pylori to some selective antibiotics and plant extracts. In analysis of information it has been used from nonparametric Cochran test and for comparisons between plant extracts of different groups, the Mcnemar and Bonferroni tests was used. In this study, bacterial viability was surveyed after being subjected to plant extracts and antibiotics by flow cytometry . Results showed that all of the bacteria were susceptible to plant extracts and the highest sensitivity was obtained with dill. All bacteria were susceptible to ciprofloxacin , tetracyclin and were resistant to amoxicillin. Flow cytometry showed that ciprofloxacin had bacteriocidal effect, tetra cyclin had bacteriostatic effect and could not kill bacteria whereas plant extracts had bacteriostatic effect .

  2. Flow cytometry studies on the Macrobrachium rosenbergii hemocytes sub-populations and immune responses to novel pathogen spiroplasma MR-1008.

    Science.gov (United States)

    Du, Jie; Zhu, Huanxi; Ren, Qian; Liu, Peng; Chen, Jing; Xiu, Yunji; Yao, Wei; Meng, Qingguo; Gu, Wei; Wang, Wen

    2012-10-01

    Flow cytometry provides rapid and reproducible methods for analyzing crustacean cellular immune responses to pathogens. We used this method to investigate the hemocytes sub-populations of freshwater prawn Macrobrachium rosenbergii and their immune responses to a novel pathogen spiroplasma MR-1008. M. rosenbergii inoculated with 100 μl spiroplasma strain MR-1008 in logarithmic phase (10(8) spiroplasmas ml(-1)) were examined for total hemocytes count (THC) and changes in differential involvement of hemocytes sub-populations during 1-28 d after inoculation. The results showed that THC was dramatically lowered 1 d after inoculation, and it obviously increased at the 5 d after inoculation; thereafter, a high level of THC was maintained to 15 d. Three morphologically distinct hemocytes sub-populations including granular cells (GC), semigranular cells (SGC) and hyaline cells (HC) could be identified by flow cytometry, and the proportions of the 3 kinds of cell categories varied obviously during the infection of spiroplasma suggesting differential involvement according to the pathogen. The flow cytometry used in this study confirmed that the semigranular cells were the main hemocytes involved in the cellular defense against spiroplasma in the M. rosenbergii.

  3. Multiplexed labeling of viable cells for high-throughput analysis of glycine receptor function using flow cytometry.

    Science.gov (United States)

    Gilbert, Daniel F; Wilson, John C; Nink, Virginia; Lynch, Joseph W; Osborne, Geoffrey W

    2009-05-01

    Flow cytometry is an important drug discovery tool because it permits high-content multiparameter analysis of individual cells. A new method dramatically enhanced screening throughput by multiplexing many discrete fixed cell populations; however, this method is not suited to assays requiring functional cellular responses. HEK293 cells were transfected with unique mutant glycine receptors. Mutant receptor expression was confirmed by coexpression of yellow fluorescent protein (YFP). Commercially available cell-permeant dyes were used to label each glycine receptor expressing mutant with a unique optical code. All encoded cell lines were combined in a single tube and analyzed on a flow cytometer simultaneously before and after the addition of glycine receptor agonist. We decoded multiplexed cells that expressed functionally distinct glycine receptor chloride channels and analyzed responses to glycine in terms of chloride-sensitive YFP expression. Here, data provided by flow cytometry can be used to discriminate between functional and nonfunctional mutations in the glycine receptor, a process accelerated by the use of multiplexing. Further, this data correlates to data generated using a microscopy-based technique. The present study demonstrates multiplexed labeling of live cells, to enable cell populations to be subject to further cell culture and experimentation, and compares the results with those obtained using live cell microscopy. (c) 2009 International Society for Advancement of Cytometry.

  4. Drug testing and flow cytometry analysis on a large number of uniform sized tumor spheroids using a microfluidic device

    Science.gov (United States)

    Patra, Bishnubrata; Peng, Chien-Chung; Liao, Wei-Hao; Lee, Chau-Hwang; Tung, Yi-Chung

    2016-02-01

    Three-dimensional (3D) tumor spheroid possesses great potential as an in vitro model to improve predictive capacity for pre-clinical drug testing. In this paper, we combine advantages of flow cytometry and microfluidics to perform drug testing and analysis on a large number (5000) of uniform sized tumor spheroids. The spheroids are formed, cultured, and treated with drugs inside a microfluidic device. The spheroids can then be harvested from the device without tedious operation. Due to the ample cell numbers, the spheroids can be dissociated into single cells for flow cytometry analysis. Flow cytometry provides statistical information in single cell resolution that makes it feasible to better investigate drug functions on the cells in more in vivo-like 3D formation. In the experiments, human hepatocellular carcinoma cells (HepG2) are exploited to form tumor spheroids within the microfluidic device, and three anti-cancer drugs: Cisplatin, Resveratrol, and Tirapazamine (TPZ), and their combinations are tested on the tumor spheroids with two different sizes. The experimental results suggest the cell culture format (2D monolayer vs. 3D spheroid) and spheroid size play critical roles in drug responses, and also demonstrate the advantages of bridging the two techniques in pharmaceutical drug screening applications.

  5. Simulation of platelets suspension flowing through a stenosis model using a dissipative particle dynamics approach.

    Science.gov (United States)

    Soares, Joao S; Gao, Chao; Alemu, Yared; Slepian, Marvin; Bluestein, Danny

    2013-11-01

    Stresses on blood cellular constituents induced by blood flow can be represented by a continuum approach down to the μm level; however, the molecular mechanisms of thrombosis and platelet activation and aggregation are on the order of nm. The coupling of the disparate length and time scales between molecular and macroscopic transport phenomena represents a major computational challenge. In order to bridge the gap between macroscopic flow scales and the cellular scales with the goal of depicting and predicting flow induced thrombogenicity, multi-scale approaches based on particle methods are better suited. We present a top-scale model to describe bulk flow of platelet suspensions: we employ dissipative particle dynamics to model viscous flow dynamics and present a novel and general no-slip boundary condition that allows the description of three-dimensional viscous flows through complex geometries. Dissipative phenomena associated with boundary layers and recirculation zones are observed and favorably compared to benchmark viscous flow solutions (Poiseuille and Couette flows). Platelets in suspension, modeled as coarse-grained finite-sized ensembles of bound particles constituting an enclosed deformable membrane with flat ellipsoid shape, show self-orbiting motions in shear flows consistent with Jeffery's orbits, and are transported with the flow, flipping and colliding with the walls and interacting with other platelets.

  6. Mechanobiology of Platelets: Techniques to Study the Role of Fluid Flow and Platelet Retraction Forces at the Micro- and Nano-Scale

    Directory of Open Access Journals (Sweden)

    Nathan J. Sniadecki

    2011-12-01

    Full Text Available Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

  7. Mechanobiology of platelets: techniques to study the role of fluid flow and platelet retraction forces at the micro- and nano-scale.

    Science.gov (United States)

    Feghhi, Shirin; Sniadecki, Nathan J

    2011-01-01

    Coagulation involves a complex set of events that are important in maintaining hemostasis. Biochemical interactions are classically known to regulate the hemostatic process, but recent evidence has revealed that mechanical interactions between platelets and their surroundings can also play a substantial role. Investigations into platelet mechanobiology have been challenging however, due to the small dimensions of platelets and their glycoprotein receptors. Platelet researchers have recently turned to microfabricated devices to control these physical, nanometer-scale interactions with a higher degree of precision. These approaches have enabled exciting, new insights into the molecular and biomechanical factors that affect platelets in clot formation. In this review, we highlight the new tools used to understand platelet mechanobiology and the roles of adhesion, shear flow, and retraction forces in clot formation.

  8. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells*

    OpenAIRE

    2009-01-01

    Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan bl...

  9. Comparison of four nuclear isolation buffers for plant DNA flow cytometry.

    Science.gov (United States)

    Loureiro, João; Rodriguez, Eleazar; Dolezel, Jaroslav; Santos, Conceição

    2006-09-01

    DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue. Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1.30-26.90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days. Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a 'fingerprint' for each buffer. The variation between days was more significant than the variation between operators. Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our

  10. Fibrocytes in the Fibrotic Lung: Altered Phenotype Detected by Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Charles eReese

    2014-06-01

    Full Text Available Fibrocytes are bone marrow hematopoietic-derived cells that also express a mesenchymal cell marker (commonly collagen I and participate in fibrotic diseases of multiple organs. Given their origin, they or their precursors must be circulating cells before recruitment into target tissues. While most previous studies focused on circulating fibrocytes, here we focus on the fibrocyte phenotype in fibrotic tissue. The study’s relevance to human disease is heightened by use of a model in which bleomycin is delivered systemically, recapitulating several features of human scleroderma including multi-organ fibrosis not observed when bleomycin is delivered directly into the lungs. Using flow cytometry, we find in the fibrotic lung a large population of CD45high fibrocytes (called Region I rarely found in vehicle-treated control mice. A second population of CD45+ fibrocytes (called Region II is observed in both control and fibrotic lung. The level of CD45 in circulating fibrocytes is far lower than in either Region I or II lung fibrocytes. The chemokine receptors CXCR4 and CCR5 are expressed at higher levels in Region I than in Region II and are present at very low levels in all other lung cells including CD45+/collagen I- leucocytes. The collagen chaperone HSP47 is present at similar high levels in both Regions I and II, but at a higher level in fibrotic lung than in control lung. There is also a major population of HSP47high/CD45- cells in fibrotic lung not present in control lung. CD44 is present at higher levels in Region I than in Region II and at much lower levels in all other cells including CD45+/collagen I- leucocytes. When lung fibrosis is inhibited by restoring caveolin-1 activity using a caveolin-1 scaffolding domain peptide (CSD, a strong correlation is observed between fibrocyte number and fibrosis score. In summary, the distinctive phenotype of fibrotic lung fibrocytes suggests that fibrocyte differentiation occurs primarily within the

  11. Flow cytometry immunophenotyping (FCI) of fine needle aspirates (FNAs) of lymph nodes.

    Science.gov (United States)

    Paro, Mirjana Mariana Kardum; Siftar, Zoran; Kardum-Skelin, Ika; Sustercić, Dunja; Nazor, Aida; Flegar-Mestrić, Zlata; Jaksić, Branimir

    2010-06-01

    Flow cytometry immunophenotyping (FCI) has an important role in the clinic work-up of fine needle aspirates (FNAs) of lymph nodes. Its standardization has been defined by proposed analytical protocols and procedures used to assure proper analytical results also in those non-routine samples. In Institute of Clinical Chemistry, "Merkur" University Hospital, FCI is accredited method according to laboratory accreditation standard ISO 15189. According to this laboratory accreditation standard, participation in external quality assessment (EQA) programs is a prerequisite for assuring integrity and quality of the entire laboratory process. A critical analysis of our institutional experience in the feasibility of FCI of the material obtained by FNA of lymph nodes with suspected lymphoma represented the purpose of the study. During an eight-year period in Institute of Clinical Chemistry, "Merkur" University Hospital, a total of 1295 FNA analysis was done, 245 of them with a possible diagnosis of B-cell Non-Hodgkin lymphomas (B-NHL) formed the basis of the study. Lymphocytes were isolated on density gradient according to Boyum et al. The average feasibility of FNAs for FCI analysis was 86% (ranged 78-93%). An acceptable total cell number in FNAs for FCI analysis (4257) was established. In total population of respondents statistical significances in expressions of cellular antigens CD3, CD5, CD22, CD23, CD19 and CD5 on B-cells (CD5+CD19+) between patient's with final diagnosis of benign, reactive lymphoid proliferations and patient's with diagnosis of B-NHL were found. EQA results analysis showed that all results were either inside target values (X +/- 1SD) or inside accepted values (X +/- 2SD). Compatibility of the restriction of immunoglobulins light chains determinated by FCI and cytomorphology diagnosis depends on the choice of criterion values of the light chains ratio which determine the monoclonality. According to the matrix of shares of all classified data of retained

  12. Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry.

    Science.gov (United States)

    de Guise, S; Flipo, D; Boehm, J R; Martineau, D; Béland, P; Fournier, M

    1995-08-01

    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.

  13. Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

    Directory of Open Access Journals (Sweden)

    Matthieu Rousseau

    Full Text Available Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(pathological processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2, comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection. This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

  14. Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

    Science.gov (United States)

    2012-01-10

    Friedrich, U. & Lenke, J. Improved Enumeration of Lactic Acid Bacteria in Mesophilic Dairy Starter Cultures by Using Multiplex Quantitative Real...messenger RNA using locked nucleic acid probes. Anal. Biochem. 390, 109-114 (2009). 13. Waters, L. & Storz, G. Regulatory RNAs in bacteria . Cell. 136, 615...Video Article Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection Kelly

  15. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

    Science.gov (United States)

    Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

    2017-01-01

    Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

  16. Photoacoustic-fluorescence in vitro flow cytometry for quantification of absorption, scattering and fluorescence properties of the cells

    Science.gov (United States)

    Nedosekin, D. A.; Sarimollaoglu, M.; Foster, S.; Galanzha, E. I.; Zharov, V. P.

    2013-03-01

    Fluorescence flow cytometry is a well-established analytical tool that provides quantification of multiple biological parameters of cells at molecular levels, including their functional states, morphology, composition, proliferation, and protein expression. However, only the fluorescence and scattering parameters of the cells or labels are available for detection. Cell pigmentation, presence of non-fluorescent dyes or nanoparticles cannot be reliably quantified. Herewith, we present a novel photoacoustic (PA) flow cytometry design for simple integration of absorbance measurements into schematics of conventional in vitro flow cytometers. The integrated system allow simultaneous measurements of light absorbance, scattering and of multicolor fluorescence from single cells in the flow at rates up to 2 m/s. We compared various combinations of excitation laser sources for multicolor detection, including simultaneous excitation of PA and fluorescence using a single 500 kHz pulsed nanosecond laser. Multichannel detection scheme allows simultaneous detection of up to 8 labels, including 4 fluorescent tags and 4 PA colors. In vitro PA-fluorescence flow cytometer was used for studies of nanoparticles uptake and for the analysis of cell line pigmentation, including genetically encoded melanin expression in breast cancer cell line. We demonstrate that this system can be used for direct nanotoxicity studies with simultaneous quantification of nanoparticles content and assessment of cell viability using a conventional fluorescent apoptosis assays.

  17. Laboratory testing for platelet function disorders.

    Science.gov (United States)

    Israels, S J

    2015-05-01

    Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories.

  18. Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

    NARCIS (Netherlands)

    Westers, T.M.; Ireland, R.; Kern, W.; Alhan, C.; Balleisen, J.S.; Bettelheim, P.; Burbury, K.; Cullen, M.; Cutler, J.A.; Porta, M.G. Della; Drager, A.M.; Feuillard, J.; Font, P.; Germing, U.; Haase, D.; Johansson, U.; Kordasti, S.; Loken, M.R.; Malcovati, L.; Marvelde, J.G. Te; Matarraz, S.; Milne, T.; Moshaver, B.; Mufti, G.J.; Ogata, K.; Orfao, A.; Porwit, A.; Psarra, K.; Richards, S.J.; Subira, D.; Tindell, V.; Vallespi, T.; Valent, P.; Velden, V.H. van der; Witte, T.J.M. de; Wells, D.A.; Zettl, F.; Bene, M.C.; Loosdrecht, A.A. van de

    2012-01-01

    Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice.

  19. A novel high-throughput multi-parameter flow cytometry based method for monitoring and rapid characterization of microbiome dynamics in anaerobic systems.

    Science.gov (United States)

    Dhoble, Abhishek S; Bekal, Sadia; Dolatowski, William; Yanz, Connor; Lambert, Kris N; Bhalerao, Kaustubh D

    2016-11-01

    A novel multidimensional flow cytometry based method has been demonstrated to monitor and rapidly characterize the dynamics of the complex anaerobic microbiome associated with perturbations in external environmental factors. While community fingerprinting provides an estimate of the meta genomic structure, flow cytometry provides a fingerprint of the community morphology including its autofluorescence spectrum in a high-throughput manner. Using anaerobic microbial consortia perturbed with the controlled addition of various carbon sources, it is possible to quantitatively discriminate between divergent microbiome analogous to community fingerprinting techniques using automated ribosomal intergenic spacer analysis (ARISA). The utility of flow cytometry based method has also been demonstrated in a fully functional industry scale anaerobic digester to distinguish between microbiome composition caused by varying hydraulic retention time (HRT). This approach exploits the rich multidimensional information from flow cytometry for rapid characterization of the dynamics of microbial communities.

  20. The Diagnostic Value of Flow Cytometry Imunophenotyping in an Albanian Patient Population with a Preliminary Clinical Diagnosis of Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Valentina Semanaj

    2014-03-01

    Conclusion: Flow cytometry immunophenotyping is a fundamental examination for the final diagnosis of chronic lymphocytic leukemia. The expression of CD38+ in CLL patients stands for a more advanced clinical stage.

  1. Microfluidic diagnostic tool for the developing world: contactless impedance flow cytometry.

    Science.gov (United States)

    Emaminejad, Sam; Javanmard, Mehdi; Dutton, Robert W; Davis, Ronald W

    2012-11-07

    In this work, we demonstrate a novel and cost-effective approach to implement a disposable microfluidic contactless impedance cytometer. Conventional methods for single cell impedance cytometry use microfabricated electrodes in direct contact with the buffer to measure changes of its electrical impedance when cells pass through the applied electric field. However, this approach requires expensive microfabrication of electrodes, and also, the fabricated electrodes cannot be reused without thorough and time-consuming cleaning process. Here, we introduce a novel approach to allow for single cell impedance cytometry using electrodes that can be reused, without the need for microfabrication of the electrodes. This disposable device can be potentially inserted onto a printed circuit board (PCB) which has a non-disposable, yet inexpensive, electronic reading apparatus. This significantly reduces the manufacturing costs, making it suitable for low resource settings, such as point-of-care testing in the developing countries.

  2. Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes-proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS.

    Science.gov (United States)

    Porwit, A; van de Loosdrecht, A A; Bettelheim, P; Brodersen, L Eidenschink; Burbury, K; Cremers, E; Della Porta, M G; Ireland, R; Johansson, U; Matarraz, S; Ogata, K; Orfao, A; Preijers, F; Psarra, K; Subirá, D; Valent, P; van der Velden, V H J; Wells, D; Westers, T M; Kern, W; Béné, M C

    2014-09-01

    Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

  3. FlowCal: A User-Friendly, Open Source Software Tool for Automatically Converting Flow Cytometry Data from Arbitrary to Calibrated Units.

    Science.gov (United States)

    Castillo-Hair, Sebastian M; Sexton, John T; Landry, Brian P; Olson, Evan J; Igoshin, Oleg A; Tabor, Jeffrey J

    2016-07-15

    Flow cytometry is widely used to measure gene expression and other molecular biological processes with single cell resolution via fluorescent probes. Flow cytometers output data in arbitrary units (a.u.) that vary with the probe, instrument, and settings. Arbitrary units can be converted to the calibrated unit molecules of equivalent fluorophore (MEF) using commercially available calibration particles. However, there is no convenient, nonproprietary tool available to perform this calibration. Consequently, most researchers report data in a.u., limiting interpretation. Here, we report a software tool named FlowCal to overcome current limitations. FlowCal can be run using an intuitive Microsoft Excel interface, or customizable Python scripts. The software accepts Flow Cytometry Standard (FCS) files as inputs and is compatible with different calibration particles, fluorescent probes, and cell types. Additionally, FlowCal automatically gates data, calculates common statistics, and produces publication quality plots. We validate FlowCal by calibrating a.u. measurements of E. coli expressing superfolder GFP (sfGFP) collected at 10 different detector sensitivity (gain) settings to a single MEF value. Additionally, we reduce day-to-day variability in replicate E. coli sfGFP expression measurements due to instrument drift by 33%, and calibrate S. cerevisiae Venus expression data to MEF units. Finally, we demonstrate a simple method for using FlowCal to calibrate fluorescence units across different cytometers. FlowCal should ease the quantitative analysis of flow cytometry data within and across laboratories and facilitate the adoption of standard fluorescence units in synthetic biology and beyond.

  4. A flow cytometry-optimized assay using an SOS-green fluorescent protein (SOS-GFP) whole-cell biosensor for the detection of genotoxins in complex environments

    DEFF Research Database (Denmark)

    Norman, Anders; Hansen, Lars H.; Sørensen, Søren Johannes

    2006-01-01

    Whole-cell biosensors have become popular tools for detection of ecotoxic compounds in environmental samples. We have developed an assay optimized for flow cytometry with detection of genotoxic compounds in mind. The assay features extended pre-incubation and a cell density of only 106-107 cells/......-contaminated soil particles when using flow cytometry, and induction of the biosensor by mitomycin C was detectable at concentrations as low as 2.5 ng/g of soil....

  5. Quantitative testing of the methodology for genome size estimation in plants using flow cytometry: a case study of the Primulina genus

    OpenAIRE

    Jing eWang; Juan eLiu; Ming eKang

    2015-01-01

    Flow cytometry (FCM) is a commonly used method for estimating genome size in many organisms. The use of flow cytometry in plants is influenced by endogenous fluorescence inhibitors and may cause an inaccurate estimation of genome size; thus, falsifying the relationship between genome size and phenotypic traits/ecological performance. Quantitative optimization of FCM methodology minimizes such errors, yet there are few studies detailing this methodology. We selected the genus Primulina, one of...

  6. Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

    Science.gov (United States)

    Żmigrodzka, M; Guzera, M; Winnicka, A

    2016-01-01

    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.

  7. High-throughput screen for the chemical inhibitors of antiapoptotic bcl-2 family proteins by multiplex flow cytometry.

    Science.gov (United States)

    Curpan, Ramona F; Simons, Peter C; Zhai, Dayong; Young, Susan M; Carter, Mark B; Bologa, Cristian G; Oprea, Tudor I; Satterthwait, Arnold C; Reed, John C; Edwards, Bruce S; Sklar, Larry A

    2011-10-01

    The human Bcl-2 family includes six antiapoptotic members (Bcl-2, Bcl-B, Bcl-W, Bcl-X(L), Bfl-1, and Mcl-1) and many proapoptotic members, wherein a balance between the two determines cell life or death in many physiological and disease contexts. Elevated expression of various antiapoptotic Bcl-2 members is commonly observed in cancers, and chemical inhibitors of these proteins have been shown to promote apoptosis of malignant cells in culture, in animal models, and in human clinical trials. All six antiapoptotic members bind a helix from the proapoptotic family member Bim, thus quenching Bim's apoptotic signal. Here, we describe the use of a multiplex, high-throughput flow cytometry assay for the discovery of small molecule modulators that disrupt the interaction between the antiapoptotic members of the Bcl-2 family and Bim. The six antiapoptotic Bcl-2 family members were expressed as glutathione-S-transferase fusion proteins and bound individually to six glutathione bead sets, with each set having a different intensity of red fluorescence. A fluorescein-conjugated Bcl-2 homology region 3 (BH3) peptide from Bim was employed as a universal ligand. Flow cytometry measured the amount of green peptide bound to each bead set in a given well, with inhibitory compounds resulting in a decrease of green fluorescence on one or more bead set(s). Hits and cheminformatically selected analogs were retested in a dose-response series, resulting in three "active" compounds for Bcl-B. These three compounds were validated by fluorescence polarization and isothermal titration calorimetry. We discuss some of the lessons learned about screening a chemical library provided by the National Institutes of Health Small Molecule Repository (∼195,000 compounds) using high-throughput flow cytometry.

  8. Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments

    Directory of Open Access Journals (Sweden)

    Munsil Wes

    2010-10-01

    Full Text Available Abstract Background The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context. Methods We apply this approach to two studies, illustrating its broad applicability. The first examines the longitudinal changes in circulating human memory T cell populations within individual patients in response to a melanoma peptide (gp100209-2M cancer vaccine, using 5 monoclonal antibodies (mAbs to delineate subpopulations of viable, gp100-specific, CD8+ T cells. The second study measures the mobilization of stem cells in porcine bone marrow that may be associated with wound healing, and uses 5 different staining panels consisting of 8 mAbs each. Results In the first study, our analysis suggests that the cell surface markers CD45RA, CD27 and CD28, commonly used in historical lower order (2-4 color flow cytometry analysis to distinguish memory from naïve and effector T cells, may not be obligate parameters in defining central memory T cells (TCM. In the second study, we identify novel phenotypes such as CD29+CD31+CD56+CXCR4+CD90+Sca1-CD44+, which may characterize progenitor cells that are significantly increased in wounded animals as compared to controls. Conclusions Taken together, these results demonstrate that Exhaustive Expansion supports thorough interrogation of complex higher-order flow cytometry data sets and aids in the identification of potentially clinically relevant findings.

  9. A flow cytometry technique to study intracellular signals NF-κB and STAT3 in peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Chavarin Patricia

    2007-07-01

    Full Text Available Abstract Background Cytokines have essential roles on intercellular communications and are effective in using a variety of intracellular pathways. Among this multitude of signalling pathways, the NF-κB (nuclear factor kappaB and STAT (signal transducer and activator of transcription families are among the most frequently investigated because of their importance. Indeed, they have important role in innate and adaptive immunity. Current techniques to study NF-κB and STAT rely on specific ELISAs, Western Blots and – most recently described – flow cytometry; so far, investigation of such signalling pathways are most commonly performed on homogeneous cells after purification. Results The present investigation aimed at developing a flow cytometry technique to study transcription factors in various cellular types such as mixtures of B-cells, T-lymphocytes and monocytes/macrophages stimulated in steady state conditions (in other words, as peripheral blood mononuclear cells. To achieve this goal, a two step procedure was carried out; the first one consisted of stimulating PBMCs with IL1β, sCD40L and/or IL10 in such a manner that optimal stimulus was found for each cell subset (and subsequent signal transduction, therefore screened by specific ELISA; the second step consisted of assessing confirmation and fine delineation of technical conditions by specific Western-Blotting for either NF-κB or STAT products. We then went on to sensitize the detection technique for mixed cells using 4 color flow cytometry. Conclusion In response to IL1β, or IL10, the levels of phosphorylated NF-κB and STAT3 – respectively – increased significantly for all the studied cell types. In contrast, B-cells and monocytes/macrophages – but, interestingly, not T-lymphocytes (in the context of PBMCs – responded significantly to sCD40L by increasing phosphorylated NF-κB.

  10. A simple multicolor flow cytometry protocol for detection and molecular characterization of circulating tumor cells in epithelial cancers.

    Science.gov (United States)

    Hristozova, Tsvetana; Konschak, Robert; Budach, Volker; Tinhofer, Ingeborg

    2012-06-01

    Circulating tumor cells (CTCs) might not only serve as prognostic marker but could also be useful for monitoring treatment efficacy. A multicolor flow cytometry protocol for their detection and molecular characterization in peripheral blood was developed which consisted of erythrocyte lysis followed by staining of cells with fluorochrome-labeled antibodies against CD45 and the epithelial markers EpCam and cytokeratin 7/8. For reducing the number of events acquired by flow cytometry, an electronic threshold for the fluorescent signals from the epithelial markers was applied. After establishment of the protocol by using spiking experiments, its suitability to determine the absolute number of CTCs as well as their expression of epidermal growth factor receptor (EGFR) and its phosphorylated form (phospho-EGFR) in blood samples from patients with squamous cell carcinoma of the head and neck (SCCHN) was validated. Spiking experiments demonstrated an excellent recovery (mean 85%) and a linear performance (R(2) = 0.98) of the protocol. Sensitivity and specificity were comparable to our former protocol using immunomagnetic CTC pre-enrichment. The analysis of 33 SCCHN patient samples revealed the presence of CTCs in 33.3% of cases with a mean ± SD of 1.5 ± 0.5 CTCs per 3.75 ml blood. EGFR was expressed in 100% and phospho-EGFR in 36.4% of the CTC+ cases. We have established a simple and sensitive multicolor flow cytometry protocol for detection of CTCs in patients with epithelial cancers including SCCHN which will allow their detailed molecular characterization.

  11. Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

    DEFF Research Database (Denmark)

    Obro, Nina F; Ryder, Lars P; Madsen, Hans O

    2012-01-01

    Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring ...

  12. Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

    Directory of Open Access Journals (Sweden)

    Tomoyuki Kaneko

    2011-06-01

    Full Text Available We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

  13. Flow Cytometry and Polymerase Chain Reaction-Based Analyses of Minimal Residual Disease in Chronic Lymphocytic Leukemia

    Directory of Open Access Journals (Sweden)

    Sabrina Uhrmacher

    2010-01-01

    Full Text Available New therapeutic strategies developed recently for chronic lymphocytic leukemia (CLL have led to remarkable treatment response rates and complete hematological remissions. This means highly sensitive and specific techniques are increasingly needed to evaluate minimal residual disease (MRD in CLL patients. Quantitative MRD levels can be used as prognostic markers, where total MRD eradication is associated with prolonged survival. Nowadays, PCR and flow cytometry techniques used to detect MRD in CLL patients can generate reliable and quantitative results with the highest sensitivity. MRD Flow is based on four-color flow cytometry using specific antibody combinations. For allele specific oligonucleotide real-time quantification (ASO RQ PCR individual primers are designed to detect a specific immunoglobulin heavy chain (IgH rearrangement in each patient clone. Five comprehensive studies investigated and compared the sensitivity and specificity of both methods. Groups of patients receiving different therapies were analyzed at different time points to generate quantitative MRD levels and MRD kinetics. All studies confirmed that both methods generate equivalent results with regard to sensitivity and MRD quantification, although each method has advantages and disadvantages in the daily routine of a standard hematological laboratory. Here, we review these investigations and compare their results in the light of modern therapies.

  14. Transcription Factors Downstream of IL-4 and TGF-β Signals: Analysis by Quantitative PCR, Western Blot, and Flow Cytometry.

    Science.gov (United States)

    Sugimoto, Atsushi; Kawakami, Ryoji; Mikami, Norihisa

    2017-01-01

    IL-9-producing Th9 cell is a novel Th cell subset involved in type II allergic inflammations such as asthma. Th9 cells can be induced from naïve Th cells in the presence of IL-4 and TGF-β. It is also well established that downstream signals of IL-4 and TGF-β, including STAT6, IRF4, Smad, and PU.1, directly mediate IL-9 production in Th9 cells. In this chapter we describe the methods of flow cytometry, qPCR and western blot analysis to determine the expression or activation of these transcription factors downstream of IL-4 and TGF-β.

  15. A Simple and Rapid Method for Quality Control of Major Histocompatibility Complex-Peptide Monomers by Flow Cytometry.

    Science.gov (United States)

    Chandran, P Anoop; Heidu, Sonja; Zelba, Henning; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Pascolo, Steve; Gouttefangeas, Cécile

    2017-01-01

    Major histocompatibility complex (MHC) multimers are essential tools in T cell immunomonitoring, which are employed both in basic and clinical research, as well as for assessing clinical samples during therapy. The generation of MHC monomers loaded with synthetic peptides is an elaborate and time-consuming process. It would be beneficial to assess the quality of these monomers prior to downstream applications. In this technical note, we describe a novel flow cytometry-based, cell-free, quick, and robust assay to check the quality of MHC monomers directly after refolding or after long-term storage.

  16. A Simple and Rapid Method for Quality Control of Major Histocompatibility Complex–Peptide Monomers by Flow Cytometry

    Science.gov (United States)

    Chandran, P. Anoop; Heidu, Sonja; Zelba, Henning; Schmid-Horch, Barbara; Rammensee, Hans-Georg; Pascolo, Steve; Gouttefangeas, Cécile

    2017-01-01

    Major histocompatibility complex (MHC) multimers are essential tools in T cell immunomonitoring, which are employed both in basic and clinical research, as well as for assessing clinical samples during therapy. The generation of MHC monomers loaded with synthetic peptides is an elaborate and time-consuming process. It would be beneficial to assess the quality of these monomers prior to downstream applications. In this technical note, we describe a novel flow cytometry-based, cell-free, quick, and robust assay to check the quality of MHC monomers directly after refolding or after long-term storage. PMID:28228758

  17. Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture.

    OpenAIRE

    McSharry, J J; Costantino, R; McSharry, M B; Venezia, R A; Lehman, J M

    1990-01-01

    Murine monoclonal antibodies (MAbs) against herpes simplex virus type 1 and 2 (HSV-1 and -2, respectively) nuclear antigens were used to identify cells infected with HSV-1 or -2 by indirect immunofluorescence in conjunction with flow cytometry after virus amplification of MRC-5 cell monolayers. The results indicate that MAbs Q1, Q2, and H-640 detect HSV-1- and HSV-2-infected cells, MAb SD-1 detects HSV-2- but not HSV-1-infected cells, and MAb 58-S detects HSV-1- but not HSV-2-infected cells. ...

  18. Selection of anticoagulant solution to the hemolymph of Chlamys farreri by transmission electron microscropy and flow cytometry

    Institute of Scientific and Technical Information of China (English)

    Chen Muyan; Yang Hongsheng; Shi Fangfang

    2007-01-01

    Mixtures of hemolymph from Chlamys farreri with three different anticoagulant solutions were incubated for an hour in vitro , then the ultrastructural alterations of hemocytes were observed , and the aggregation rate was analyzed by using transmission electron microscropy and flow cytometry respectively. The results showed that Formula 3 (glucose 20. 8 g L-1; EDTA 20mM ; sodium chloride 20 g L-1 ; Tris-HCl 0.05M;pH 7. 4) was the desirable anticoagulant solution for C . Farreri hemocytes. Further phagocytosis assay showed that no obvious negative effect was given to the hemocyte phagocytic activity when using Formula 3 as the anticoagulant solution.

  19. Microflow1, a sheathless fiber-optic flow cytometry biomedical platform: demonstration onboard the international space station.

    Science.gov (United States)

    Dubeau-Laramée, Geneviève; Rivière, Christophe; Jean, Isabelle; Mermut, Ozzy; Cohen, Luchino Y

    2014-04-01

    A fiber-optic based flow cytometry platform was designed to build a portable and robust instrument for space applications. At the core of the Microflow1 is a unique fiber-optic flow cell fitted to a fluidic system and fiber coupled to the source and detection channels. A Microflow1 engineering unit was first tested and benchmarked against a commercial flow cytometer as a reference in a standard laboratory environment. Testing in parabolic flight campaigns was performed to establish Microflow1's performance in weightlessness, before operating the new platform on the International Space Station. Microflow1 had comparable performances to commercial systems, and operated remarkably and robustly in weightlessness (microgravity). Microflow1 supported immunophenotyping as well as microbead-based multiplexed cytokine assays in the space environment and independently of gravity levels. Results presented here provide evidence that this fiber-optic cytometer technology is inherently compatible with the space environment with negligible compromise to analytical performance.

  20. On Weak Plane Couette and Poiseuille Flows of Rigid Rod and Platelet Ensembles

    Science.gov (United States)

    2006-01-01

    SIAM J. APPL. MATH. c© 2006 Society for Industrial and Applied Mathematics Vol. 66, No. 4, pp. 1227–1260 ON WEAK PLANE COUETTE AND POISEUILLE FLOWS ...anisotropic elasticity; to compare Couette versus Poiseuille flow ; and to consider dynamics and stability of these steady states within the asymptotic model...On Weak Plane Couette and Poiseuille Flows of Rigid Rod and Platelet Ensembles 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6

  1. Effect of swirling flow on platelet concentration distribution in small-caliber artificial grafts and end-to-end anastomoses

    Institute of Scientific and Technical Information of China (English)

    Fan Zhan; Yu-Bo Fan; Xiao-Yan Deng

    2011-01-01

    Platelet concentration near the blood vessel wall is one of the major factors in the adhesion of platelets to the wall.In our previous studies,it was found that swirling flows could suppress platelet adhesion in small-caliber artificial grafts and end-to-end anastomoses.In order to better understand the beneficial effect of the swirling flow,we numerically analyzed the near-wall concentration distribution of platelets in a straight tube and a sudden tubular expansion tube under both swirling flow and normal flow conditions.The numerical models were created based on our previous experimental studies.The simulation results revealed that when compared with the normal flow,the swirling flow could significantly reduce the near-wall concentration of platelets in both the straight tube and the expansion tube.The present numerical study therefore indicates that the reduction in platelet adhesion under swirling flow conditions in small-caliber arterial grafts,or in end-to-end anastomoses as observed in our previous experimental study,was possibly through a mechanism of platelet transport,in which the swirling flow reduced the near-wall concentration of platelets.

  2. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

    Directory of Open Access Journals (Sweden)

    Zheng Zhili

    2012-02-01

    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

  3. Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

    Science.gov (United States)

    Adams, Rebecca L C; Cheung, Catherine; Banh, Raymond; Saal, Russell; Cross, Donna; Gill, Devinder; Self, Marlene; Klein, Kerenaftali; Mollee, Peter

    2014-03-01

    Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). By comparing three different flow cytometry methods using receiver–operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients. © 2013 International Clinical Cytometry Society.

  4. Identification of residual leukemic cells by flow cytometry in childhood B-cell precursor acute lymphoblastic leukemia: verification of leukemic state by flow-sorting and molecular/cytogenetic methods

    OpenAIRE

    2012-01-01

    Reduction in minimal residual disease, measured by real-time quantitative PCR or flow cytometry, predicts prognosis in childhood B-cell precursor acute lymphoblastic leukemia. We explored whether cells reported as minimal residual disease by flow cytometry represent the malignant clone harboring clone-specific genomic markers (53 follow-up bone marrow samples from 28 children with B-cell precursor acute lymphoblastic leukemia). Cell populations (presumed leukemic and non-leukemic) were flow-s...

  5. [THE NEW APPROACH TO EVALUATION OF ENDOTHELIUM DYSFUNCTION: DETECTION OF NUMBER OF CIRCULATING ENDOTHELIUM CELLS USING FLOW CYTOMETRY TECHNIQUE].

    Science.gov (United States)

    Feoktistova, V S; Vavilkova, T V; Sirotkina, O V; Boldueva, S A; Gaikovaia, L B; Leonova, I A; Laskovets, A B; Ermakov, A I

    2015-04-01

    The endothelium dysfunction takes leading place in pathogenesis of development of cardiovascular diseases. The circulating endothelium cells of peripheral blood can act as a direct cell marker of damage and remodeling of endothelium. The study was carried out to develop a new approach to diagnose of endothelium dysfunction by force of determination of number of circulating endothelium cells using flow cytometry technique and to apply determination of circulating endothelium cells for evaluation of risk of development of ischemic heart disease in women of young and middle age. The study embraced 62 female patients with angiography confirmed ischemic heart disease, exertional angina pectoris at the level of functional class I-II (mean age 51 ± 6 years) and 49 women without anamnesis of ischemic heart disease (mean age 52 ± 9 years). The occurrence of more than three circulating endothelium cells by 3 x 105 leukocytes in peripheral blood increases relative risk of development of ischemic heart disease up to 4 times in women of young and middle age and risk of development of acute myocardial infarction up to 8 times in women with ischemic heart disease. The study demonstrated possibility to apply flow cytometry technique to quantitatively specify circulating endothelium cells in peripheral blood and forecast risk of development of ischemic heart disease in women of young and middle age depending on level of circulating endothelium cells.

  6. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

    Science.gov (United States)

    Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

    2015-12-01

    Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

  7. Flow cytometry analysis of hormone receptors on human peripheral blood mononuclear cells to identify stress-induced neuroendocrine effects

    Science.gov (United States)

    Meehan, R. T.

    1986-01-01

    Understanding the role of circulating peptide hormones in the pathogenesis of space-flight induced disorders would be greatly facilitated by a method which monitors chronic levels of hormones and their effects upon in vivo cell physiology. Single and simultaneous multiparameter flow cytometry analysis was employed to identify subpopulations of mononuclear cells bearing receptors for ACTH, Endorphin, and Somatomedin-C using monoclonal antibodies and monospecific antisera with indirect immunofluorescence. Blood samples were obtained from normal donors and subjects participating in decompression chamber studies (acute stress), medical student academic examination (chronic stress), and a drug study (Dexamethasone). Preliminary results indicate most ACTH and Endorphin receptor positive cells are monocytes and B-cells, exhibit little diurnal variation but the relative percentages of receptor positive cells are influenced by exposure to various stressors and ACTH inhibition. This study demonstrates the capability of flow cytometry analysis to study cell surface hormone receptor regulation which should allow insight into neuroendocrine modulation of the immune and other cellular systems during exposure to stress or microgravity.

  8. Circulating plasma cells detected by flow cytometry as a predictor of survival in 302 patients with newly diagnosed multiple myeloma.

    Science.gov (United States)

    Nowakowski, Grzegorz S; Witzig, Thomas E; Dingli, David; Tracz, Michal J; Gertz, Morie A; Lacy, Martha Q; Lust, John A; Dispenzieri, Angela; Greipp, Philip R; Kyle, Robert A; Rajkumar, S Vincent

    2005-10-01

    We detected circulating plasma cells (PCs) by flow cytometry in 302 patients with newly diagnosed multiple myeloma (MM) by gating on CD38+CD45- cells. The number of circulating PCs per 50 000 mononuclear cells was reported. In 80 (27%) patients, no circulating PC were seen; 106 (35%) patients had 1 to 10 and 115 (38%) patients had more than 10 circulating PCs. Median overall survival for the 302 patients was 47 months. Patients with 10 or fewer circulating PCs had a median survival of 58.7 months, whereas patients with more than 10 circulating PCs had a median survival of 37.3 months (P = .001). On multivariate analysis, the prognostic value of circulating PCs was independent of beta2-microglobulin, albumin, and C-reactive protein. There was only a weak correlation between tumor mass and circulating PCs, suggesting that the appearance of circulating PCs may be a reflection of tumor biology. We conclude that the number of circulating PCs measured by flow cytometry in patients with newly diagnosed MM is an independent predictor of survival.

  9. Multiparametric flow cytometry allows rapid assessment and comparison of lactic acid bacteria viability after freezing and during frozen storage.

    Science.gov (United States)

    Rault, Aline; Béal, Catherine; Ghorbal, Sarrah; Ogier, Jean-Claude; Bouix, Marielle

    2007-08-01

    Freezing is widely used for the long-term preservation of lactic acid bacteria, but often affects their viability and technological properties. Different methods are currently employed to determine bacterial cryotolerance, but they all require several hours or days before achieving results. The aim of this study was to establish the advantages of multiparametric flow cytometry by using two specific fluorescent probes to provide rapid assessment of the viability of four strains of Lactobacillus delbrueckii after freezing and during frozen storage. The relevance of carboxyfluorescein diacetate and propidium iodide to quantify bacterial viability was proven. When bacterial suspensions were simultaneously stained with these two fluorescent probes, three major subpopulations were identified: viable, dead and injured cells. The cryotolerance of four L. delbrueckii strains was evaluated by quantifying the relative percentages of each subpopulation before and after freezing, and throughout one month of storage at -80 degrees C. Results displayed significant differences in the resistance to freezing and frozen storage of the four strains when they were submitted to the same freezing and storage procedures. Whereas resistant strains displayed less than 10% of dead cells after one month of storage, one sensitive strain exhibited more than 50% of dead cells, together with 14% of stressed cells after freezing. Finally, this study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of lactic acid bacteria, and was well correlated with plate count results. Moreover, it made it possible to differentiate strains according to their susceptibility to freezing and frozen storage.

  10. Flow-cytometry and cell sorting: an efficient approach to investigate productivity and cell physiology in mammalian cell factories.

    Science.gov (United States)

    Kumar, Niraj; Borth, Nicole

    2012-03-01

    The performance of cell lines used for the production of biotherapeutic proteins typically depends on the number of cells in culture, their specific growth rate, their viability and the cell specific productivity (qP). Therefore both cell line development and process development are trying to (a) improve cell proliferation to reduce lag-phase and achieve high number of cells; (b) delay cell death to prolong the production phase and improve culture longevity; (c) and finally, increase qP. All of these factors, when combined in an optimised process, concur to increase the final titre and yield of the recombinant protein. As cellular performance is at the centre of any improvement, analysis methods that enable the characterisation of individual cells in their entirety can help in identifying cell types and culture conditions that perform exceptionally well. This observation of cells and their complexity is reflected by the term "cytomics" and flow cytometry is one of the methods used for this purpose. With its ability to analyse the distribution of physiological properties within a population and to isolate rare outliers with exceptional properties, flow cytometry ideally complements other methods used for optimisation, including media design and cell engineering. In the present review we describe approaches that could be used, directly or indirectly, to analyse and sort cellular phenotypes characterised by improved growth behaviour, reduced cell death or high qP and outline their potential use for cell line and process optimisation.

  11. Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

    Science.gov (United States)

    Clavarino, Giovanna; Delouche, Noémie; Vettier, Claire; Laurin, David; Pernollet, Martine; Raskovalova, Tatiana; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal; Jacob, Marie-Christine

    A precise identification and phenotypic characterization of human B-cell subsets is of crucial importance in both basic research and medicine. In the literature, flow cytometry studies for the phenotypic characterization of B-lymphocytes are mainly focused on the description of a particular cell stage, or of specific cell stages observed in a single type of sample. In the present work, we propose a backbone of 6 antibodies (CD38, CD27, CD10, CD19, CD5 and CD45) and an efficient gating strategy to identify, in a single analysis tube, a large number of B-cell subsets covering the whole B-cell differentiation from precursors to memory and plasma cells. Furthermore, by adding two antibodies in an 8-color combination, our approach allows the analysis of the modulation of any cell surface marker of interest along B-cell differentiation. We thus developed a panel of seven 8-colour antibody combinations to phenotypically characterize B-cell subpopulations in bone marrow, peripheral blood, lymph node and cord blood samples. Beyond qualitative information provided by biparametric representations, we also quantified antigen expression on each of the identified B-cell subsets and we proposed a series of informative curves showing the modulation of seventeen cell surface markers along B-cell differentiation. Our approach by flow cytometry provides an efficient tool to obtain quantitative data on B-cell surface markers expression with a relative easy-to-handle technique that can be applied in routine explorations.

  12. Notes on genome size in the hybrid Ranunculus x luizetii (Ranunculaceae and its parents by flow cytometry

    Directory of Open Access Journals (Sweden)

    Fernández Prieto, J. A.

    2011-12-01

    Full Text Available Notes on genome size in the hybrid Ranunculus x luizetii (Ranunculaceae and its parents by flow cytometry.- Flow cytometry was used to estimate the nuclear DNA content in the natural hybrid Ranunculus x luizetii and its parents. Our results indicate that the genome size of the hybrid R. x luizetii is closer to R. pyrenaeus than to R. parnassiifolius, providing an evidence of genome downsizing.Notas sobre el tamaño del genoma en el híbrido Ranunculus x luizetii (Ranunculaceae y sus progenitores mediante citometría de flujo.- Se ha empleado la citometría de flujo para estimar el contenido de ADN nuclear en el híbrido Ranunculus x luizetii y sus progenitores. Nuestros resultados indican que el tamaño del genoma del híbrido R. x luizetii se acerca más a R. pyrenaeus que a R. parnassiifolius, con una evidencia de reducción del genoma.

  13. Imaging Flow Cytometry for Multiparametric Analysis of Molecular Mechanism Involved in the Cytotoxicity of Human CD8(+) T-cells.

    Science.gov (United States)

    Wabnitz, Guido H; Kirchgessner, Henning; Samstag, Yvonne

    2017-09-01

    The clearance of tumors or virus infected cells is a crucial task of the immune system. Cytotoxic T-cells (CTLs) are able to detect and to kill such altered host cells. Given the recent success of checkpoint inhibitors for tumor therapy, it becomes more and more important to understand the biology of T-cell mediated target cell killing. Tests that allow analyzing the biology of CTLs are either based on flow cytometry or fluorescence microscopy. Thus, they either lack image-based information or have a poor statistical robustness. Therefore, we describe an approach to quantify CTL-mediated cytotoxicity using imaging flow cytometry. Using activated primary human cytotoxic T-cells as CTLs and P815 as target cells, we show that both the evaluation of target cell death and the biology of CTLs can be evaluated in parallel. This enables to gain information about CTL-mediated cytotoxicity in samples from patients important for translational medicine. J. Cell. Biochem. 118: 2528-2533, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics

    Science.gov (United States)

    Sgier, Linn; Freimann, Remo; Zupanic, Anze; Kroll, Alexandra

    2016-01-01

    Biofilms serve essential ecosystem functions and are used in different technical applications. Studies from stream ecology and waste-water treatment have shown that biofilm functionality depends to a great extent on community structure. Here we present a fast and easy-to-use method for individual cell-based analysis of stream biofilms, based on stain-free flow cytometry and visualization of the high-dimensional data by viSNE. The method allows the combined assessment of community structure, decay of phototrophic organisms and presence of abiotic particles. In laboratory experiments, it allows quantification of cellular decay and detection of survival of larger cells after temperature stress, while in the field it enables detection of community structure changes that correlate with known environmental drivers (flow conditions, dissolved organic carbon, calcium) and detection of microplastic contamination. The method can potentially be applied to other biofilm types, for example, for inferring community structure for environmental and industrial research and monitoring. PMID:27188265

  15. A flow cytometry-based workflow for detection and quantification of anti-plasmodial antibodies in vaccinated and naturally exposed individuals

    DEFF Research Database (Denmark)

    Ajua, Anthony; Engleitner, Thomas; Esen, Meral;

    2012-01-01

    information about natural exposure and vaccine immunogenicity. A novel, cytometry-based workflow for the quantitative detection of anti-plasmodial antibodies in human serum is presented. METHODS: Fixed red blood cells (RBCs), infected with late stages of P. falciparum were utilized to detect malaria......-specific antibodies by flow cytometry with subsequent automated data analysis. Available methods for data-driven analysis of cytometry data were assessed and a new overlap subtraction algorithm (OSA) based on open source software was developed. The complete workflow was evaluated using sera from two GMZ2 malaria...... vaccine trials in semiimmune adults and pre-school children residing in a malaria endemic area. RESULTS: Fixation, permeabilization, and staining of infected RBCs were adapted for best operation in flow cytometry. As asexual vaccine candidates are designed to induce antibody patterns similar to semi...

  16. Time-domain microfluidic fluorescence lifetime flow cytometry for high-throughput Förster resonance energy transfer screening.

    Science.gov (United States)

    Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

    2015-02-01

    Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5-5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence

  17. Platelet antibodies, activated platelets and serum leptin in childhood immune thrombocytopenic purpura.

    Science.gov (United States)

    Badrawy, Hosny; Elsayh, Khalid I; Zahran, Asmaa M; El-Ghazali, Mohamad Hamdy

    2013-01-01

    The aim of this study was to evaluate the levels of platelet-associated antibodies (PAIgG and PAIgM), activated platelets and serum leptin in children with acute immune thrombocytopenic purpura (ITP). The study included 40 patients with ITP and 40 healthy age- and sex-matched controls. PAIgG, PAIgM and activated platelet levels were estimated by flow cytometry, and serum leptin levels were estimated by ELISA. Activated platelets and serum leptin were significantly higher in the ITP patients than in the controls. The percentage and mean fluorescence intensity of PAIgG and PAIgM staining were significantly higher in the patients than in the controls. Serum leptin and activated platelet levels in patients with thrombocytopenia of brief duration were significantly lower than those in patients with thrombocytopenia of prolonged duration. The levels of activated platelets, serum leptin and PAIgG were positively correlated, and the levels of serum leptin, activated platelets and platelet counts were negatively correlated. The increased levels of activated platelets, serum leptin and platelet-associated antibodies in children with acute ITP suggest that these factors could play a role in ITP pathogenesis. Additionally, activated platelets and serum leptin could have prognostic significance in paediatric acute ITP. Copyright © 2013 S. Karger AG, Basel.

  18. Effects of methylprednisolone on concanavalin A-induced human lymphocyte blastogenesis: a comparative analysis by flow cytometry, volume determination and /sup 3/H-thymidine incorporation

    Energy Technology Data Exchange (ETDEWEB)

    Marder, P.; Schmidtke, J.R.

    1983-08-01

    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.

  19. Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

    Science.gov (United States)

    Shahrokh Esfahani, Samaneh; Emtiazi, Giti; Shafiei, Rasoul; Ghorbani, Najmeh; Zarkesh Esfahani, Seyed Hamid

    2016-09-01

    The Bacillus species have many applications in the preparation of various enzymes, probiotic, biofertilizer, and biomarkers for which the survival of resting cells and spore formation under different conditions are important. In this study, water and saline along with different mineral substances such as calcium carbonate, calcium phosphate, and silica were used for the detection of survival and preservation of Bacillus amyloliquefaciens. The results showed intensive death of resting cells at 8 °C, but significant survival at 28 °C after one month. However, preservation by minerals significantly decreased the rate of death and induced sporulation at both the temperatures. The resting cells were maintained at room temperature (about 60 % of the initial population survived after a month) in the presence of tricalcium phosphate. The results showed that temperature has more effect on sporulation compare with starvation. The sporulation in normal saline at 28 °C was 70 times more than that at 8 °C; meanwhile, addition of tricalcium phosphate increases sporulation by 90 times. Also, the FTIR data showed the interaction of tricalcium phosphate with spores and resting cells. The discrimination of sporulation from non-sporulation state was performed by nucleic acid staining with thiazole orange and detected by flow cytometry. The flow cytometric studies confirmed that the rates of sporulation in pure water were significantly more at 28 °C. This is the first report on the detection of bacterial spore with thiazole orange by flow cytometry and also on the interaction of tricalcium phosphate with spores by FTIR analyses.

  20. Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.

    Science.gov (United States)

    Grimberg, Brian T; Erickson, John J; Sramkoski, R Michael; Jacobberger, James W; Zimmerman, Peter A

    2008-06-01

    The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy. (c) 2008 International Society for Advancement of Cytometry.

  1. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

    Science.gov (United States)

    Bell, D N; Spain, S; Goldsmith, H L

    1989-11-01

    A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.

  2. Analysis of Flow Cytometry DNA Damage Response Protein Activation Kinetics Following X-rays and High Energy Iron Nuclei Exposure

    Energy Technology Data Exchange (ETDEWEB)

    Universities Space Research Association; Chappell, Lori J.; Whalen, Mary K.; Gurai, Sheena; Ponomarev, Artem; Cucinotta, Francis A.; Pluth, Janice M.

    2010-12-15

    We developed a mathematical method to analyze flow cytometry data to describe the kinetics of {gamma}H2AX and pATF2 phosphorylations ensuing various qualities of low dose radiation in normal human fibroblast cells. Previously reported flow cytometry kinetic results for these DSB repair phospho-proteins revealed that distributions of intensity were highly skewed, severely limiting the detection of differences in the very low dose range. Distributional analysis reveals significant differences between control and low dose samples when distributions are compared using the Kolmogorov-Smirnov test. Radiation quality differences are found in the distribution shapes and when a nonlinear model is used to relate dose and time to the decay of the mean ratio of phosphoprotein intensities of irradiated samples to controls. We analyzed cell cycle phase and radiation quality dependent characteristic repair times and residual phospho-protein levels with these methods. Characteristic repair times for {gamma}H2AX were higher following Fe nuclei as compared to X-rays in G1 cells (4.5 {+-} 0.46 h vs 3.26 {+-} 0.76 h, respectively), and in S/G2 cells (5.51 {+-} 2.94 h vs 2.87 {+-} 0.45 h, respectively). The RBE in G1 cells for Fe nuclei relative to X-rays for {gamma}H2AX was 2.05 {+-} 0.61 and 5.02 {+-} 3.47, at 2 h and 24-h postirradiation, respectively. For pATF2, a saturation effect is observed with reduced expression at high doses, especially for Fe nuclei, with much slower characteristic repair times (>7 h) compared to X-rays. RBEs for pATF2 were 0.66 {+-} 0.13 and 1.66 {+-} 0.46 at 2 h and 24 h, respectively. Significant differences in {gamma}H2AX and pATF2 levels comparing irradiated samples to control were noted even at the lowest dose analyzed (0.05 Gy) using these methods of analysis. These results reveal that mathematical models can be applied to flow cytometry data to uncover important and subtle differences following exposure to various qualities of low dose radiation.

  3. Platelet function tests: a comparative review

    Directory of Open Access Journals (Sweden)

    Paniccia R

    2015-02-01

    Full Text Available Rita Paniccia,1,2 Raffaella Priora,1,2 Agatina Alessandrello Liotta,2 Rosanna Abbate1,2 1Department of Experimental and Clinical Medicine, Thrombosis Center, University of Florence, Florence, Italy; 2Department of Heart and Vessels, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy Abstract: In physiological hemostasis a prompt recruitment of platelets on the vessel damage prevents the bleeding by the rapid formation of a platelet plug. Qualitative and/or quantitative platelet defects promote bleeding, whereas the high residual reactivity of platelets in patients on antiplatelet therapies moves forward thromboembolic complications. The biochemical mechanisms of the different phases of platelet activation – adhesion, shape change, release reaction, and aggregation – have been well delineated, whereas their complete translation into laboratory assays has not been so fulfilled. Laboratory tests of platelet function, such as bleeding time, light transmission platelet aggregation, lumiaggregometry, impedance aggregometry on whole blood, and platelet activation investigated by flow cytometry, are traditionally utilized for diagnosing hemostatic disorders and managing patients with platelet and hemostatic defects, but their use is still limited to specialized laboratories. To date, a point-of-care testing (POCT dedicated to platelet function, using pertinent devices much simpler to use, has now become available (ie, PFA-100, VerifyNow System, Multiplate Electrode Aggregometry [MEA]. POCT includes new methodologies which may be used in critical clinical settings and also in general laboratories because they are rapid and easy to use, employing whole blood without the necessity of sample processing. Actually, these different platelet methodologies for the evaluation of inherited and acquired bleeding disorders and/or for monitoring antiplatelet therapies are spreading and the study of platelet function is strengthening. In this review, well

  4. Platelet activation patterns in platelet size sub-populations: differential responses to aspirin in vitro.

    Science.gov (United States)

    Mangalpally, Kiran Kumar R; Siqueiros-Garcia, Alan; Vaduganathan, Muthiah; Dong, Jing-Fei; Kleiman, Neal S; Guthikonda, Sasidhar

    2010-10-01

    Circulating platelets are heterogeneous in size and structure. Whether this translates into differences in platelet function and efficacy of antiplatelet therapy is unclear. Hence, we decided to investigate the activation patterns among different platelet populations differentiated by size, and to compare the inhibitory effects of aspirin in these populations. Circulating platelets from 9 healthy volunteers were separated by size and stratified into the largest and smallest quintiles. Platelets were stimulated with 75 μM arachidonic acid (AA), 10 μM ADP or 25 μM TRAP. Alpha-granule protein secretion and expression (P-selectin, VWF, fibrinogen), surface-protein activation (activated integrin αIIbβ3) were assessed. Platelet thromboxane B(2) (TxB(2)) synthesis following AA stimulation was measured in vitro before and after incubation with 265 μM aspirin. Reticulated (juvenile) platelets were assessed using thiazole orange staining. A greater number of large platelets in the largest quintile were reticulated compared with the smallest quintile (6.1 ± 2.8% vs. 1.2 ± 1.5% respectively, p aspirin (1029 ± 190 pg/mL vs. 851 ± 159 pg/mL, respectively, p = 0.03). After stimulation with each agonist, a greater proportion of large platelets bound fibrinogen, VWF, P-selectin and activated integrin αIIbβ3 than small platelets both in the presence and in the absence of in vitro aspirin. In an in vitro setting, large platelets appear to be more active than small platelets and continue to be more active even after in vitro aspirin. Platelets exhibit heterogeneity in size and structure. Whether this translates into platelet function and efficacy of antiplatelet therapy is unclear. We evaluated platelet functional properties and the effects of aspirin on separated platelet subpopulations in an in vitro setting. Platelets were sorted into the largest and smallest size quintiles using flow cytometry forward scatter. Alpha-granule protein release, dense granule content

  5. Experimental simulation of model platelet adhesion to a semi-permeable wall exposed to flow disturbance

    Institute of Scientific and Technical Information of China (English)

    DENG Xiaoyan; WANG Guixue; YANG Yang

    2003-01-01

    A sudden tubular expansion with a semi- permeable wall was constructed from a tubular dialysis membrane to investigate the effects of filtration flow and flow disturbance on particle deposition. The expansion was perfused with a dilute, neutrally buoyant suspension of 1.10 ?m diameter polystyrene latex spheres (as models of platelets) in Tris buffer solution containing 10% Dextran T70 and 2% bovine serum albumin. The results showed that adhesion of particles correlated positively with the filtration rate and inversely with the wall shear rate. In the vortex flow region distal to the expansion, particle adhesion was significantly elevated with a maximum at the reattachment point where the wall shear rate was the lowest and particles were constantly carried toward the vessel wall along the curved streamlines. In conclusion, filtration flow has a profound impact on the interaction of blood cells such as platelets with blood vessel walls, and the disturbed flow with a low wall shear rate can enhance the deposits of platelet thrombi to the vessel wall.

  6. Flow cytometry in environmental microbiology: a rapid approach for the isolation of single cells for advanced molecular biology analysis.

    Science.gov (United States)

    Ferrari, Belinda C; Winsley, Tristrom J; Bergquist, Peter L; Van Dorst, Josie

    2012-01-01

    The isolation and subsequent characterization of microbial cells from within environmental samples is a difficult process. Flow cytometry and cell sorting, when combined with the application of fluorescent probes, have the capability for the detection and separation of diverse microbial populations from within complex mixtures. The isolation of single cells allows for downstream investigations towards system-level characterization of unknown Bacterial Phyla to occur. We describe here the combination of fluorescent in situ hybridization and cell sorting for the detection and isolation of Candidate Division TM7 bacteria from an enriched soil sample. The result is the isolation of rare cells suitable for advanced molecular analysis including whole genome amplification and high-throughput pyrosequencing.

  7. [The application of method of flow cytometry in diagnostics of hereditary spherocytosis (eosin-5 maleimid binding test)].

    Science.gov (United States)

    Prokhorova, Iu A; Zueva, E E; Sokolova, N E

    2012-07-01

    The technique of binding eosin-5 maleimid fluorescent dye with lysine-430 of first extracellular protein bulge of band 3 of erythrocytes' membranes makes it possible to detect the defects of cytoskeleton of erythrocytes as a biological foundation of pathogenesis of hereditary spherocytosis. The samples of peripheral blood from 125 adult persons and 18 children with established absence of hematologic disorders were analyzed The samples of peripheral blood from 19 patients with verified hereditary spherocytosis were analyzed too. The method of flow cytometry was applied to register the average intensity of fluorescence of eosin-5 maleimid. The decrease of average intensity of fluorescence of eosin-5 maleimid of erythrocytes of patients with hereditary spherocytosis as compared with data from comparison groups was established in all cases.

  8. Flow Cytometry Method Analysis of Apoptosis: No Significant Difference Between EDTA and EDTA-free Trypsin Treatment Procedure.

    Science.gov (United States)

    Xu, Xiao-yan; Nie, Xiao-cui; Ma, Hai-ying; Song, Guo-qing; Zhang, Xiao-tong; Jin, Yu-nan; Yu, Yan-qiu

    2015-04-01

    Flow cytometry method (FCM) is a generally accepted tool to analyze apoptosis. Although apoptosis assay kit was applied by many companies, the manufacturers were not consistent with whether using Trypsin with EDTA to collect the adherent cells. In another words, the influence of EDTA on apoptotic ratio is not clear. In this work, we compared the proportion of apoptotic cells with EDTA or EDTA-free Trypsin treatment by FCM. We concluded that Trypsin with or without EDTA has little influence on the proportion of apoptotic cells. In addition, we found that the ratio of necrosis and apoptosis was different in cells collected by scraping. WAVE2 protein was analyzed as a typical example for movement related protein. WAVE2 expression is elevated in the EDTA Trypsin treated group, compared with EDTA-free Trypsin treatment and scrapping group.

  9. A quantitative method for measurement of HL-60 cell apoptosis based on diffraction imaging flow cytometry technique.

    Science.gov (United States)

    Yang, Xu; Feng, Yuanming; Liu, Yahui; Zhang, Ning; Lin, Wang; Sa, Yu; Hu, Xin-Hua

    2014-07-01

    A quantitative method for measurement of apoptosis in HL-60 cells based on polarization diffraction imaging flow cytometry technique is presented in this paper. Through comparative study with existing methods and the analysis of diffraction images by a gray level co-occurrence matrix algorithm (GLCM), we found 4 GLCM parameters of contrast (CON), cluster shade (CLS), correlation (COR) and dissimilarity (DIS) exhibit high sensitivities as the apoptotic rates. It was further demonstrated that the CLS parameter correlates significantly (R(2) = 0.899) with the degree of nuclear fragmentation and other three parameters showed a very good correlations (R(2) ranges from 0.69 to 0.90). These results demonstrated that the new method has the capability for rapid and accurate extraction of morphological features to quantify cellular apoptosis without the need for cell staining.

  10. Assessment of morphological changes of Clostridium acetobutylicum by flow cytometry during acetone/butanol/ethanol extractive fermentation.

    Science.gov (United States)

    González-Peñas, Helena; Lu-Chau, Thelmo Alejandro; Moreira, Maria Teresa; Lema, Juan Manuel

    2015-03-01

    Acetone/butanol/ethanol (ABE) fermentation by Clostridium acetobutylicum was investigated in extractive fed-batch experiments. In conventional fermentations, metabolic activity ceases when a critical threshold products concentration is reached (~21.6 g solvents l(-1)). Solvents production was increased up to 36.6 and 37.2 g l(-1), respectively, using 2-butyl-1-octanol (aqueous to organic ratio: 1:0.25 v/v) and pomace olive oil (1:1 v/v) as extraction solvents. The morphological changes of different cell types were monitored and quantified using flow cytometry. Butanol production in extractive fermentations with pomace olive oil was achieved mainly by vegetative cells, whereas the percentage of sporulating cells was lower than 10%.

  11. Precise determinations of C and D periods by flow cytometry in Escherichia coli K-12 and B/r

    DEFF Research Database (Denmark)

    Michelsen, Ole; de Mattos, M.J.T.; Jensen, Peter Ruhdal;

    2003-01-01

    The C and D cell cycle periods of seven Escherichia coli K-12 strains and three E, coli B/r strains were determined by computer simulation of DNA histograms obtained by flow cytometry of batch cultures grown at several different generation times. To obtain longer generation times two of the K-12...... strains were cultivated at several different dilution rates in glucose-limited chemostats. The replication period (C period) was found to be similar in K-12 and B/r strains grown at similar generation times. At generation times below 60 min the C period was constant; above 60 min it increased linearly...... with increasing generation time. The period from termination of replication to cell division (D period) was more variable. It was much shorter in B/r than in K-12 strains. Like the C period it was relatively constant at generation times below 60 min and it increased with increasing generation times at longer...

  12. Multi-parameter flow cytometry and cell sorting reveal extensive physiological heterogeneity in Bacillus cereus batch cultures.

    Science.gov (United States)

    Want, Andrew; Hancocks, Helen; Thomas, Colin R; Stocks, Stuart M; Nebe-von-Caron, Gerhard; Hewitt, Christopher J

    2011-07-01

    Based on two staining protocols, DiOC(6)(3)/propidium iodide (PI) and RedoxSensor Green (an indicator of bacterial reductase activity)/PI, multi-parameter flow cytometry and cell sorting has identified at least four distinguishable physiological states during batch cultures of Bacillus cereus. Furthermore, dependent on the position in the growth curve, single cells gave rise to varying numbers of colonies when sorted individually onto nutrient agar plates. These growing colonies derived from a single cell had widely different lag phases, inferred from differences in colony size. This further highlights the complex population dynamics of bacterial monocultures and further demonstrates that individual bacterial cells in a culture respond in markedly dissimilar ways to the environment, resulting in a physiologically heterogenous and dynamic population.

  13. Coriander (Coriandrum sativum L.) essential oil: its antibacterial activity and mode of action evaluated by flow cytometry.

    Science.gov (United States)

    Silva, Filomena; Ferreira, Susana; Queiroz, João A; Domingues, Fernanda C

    2011-10-01

    The aim of this work was to study the antibacterial effect of coriander (Coriandrum sativum) essential oil against Gram-positive and Gram-negative bacteria. Antibacterial susceptibility was evaluated using classical microbiological techniques concomitantly with the use of flow cytometry for the evaluation of cellular physiology. Our results showed that coriander oil has an effective antimicrobial activity against all bacteria tested. Also, coriander oil exhibited bactericidal activity against almost all bacteria tested, with the exception of Bacillus cereus and Enterococcus faecalis. Propidium iodide incorporation and concomitant loss of all other cellular functions such as efflux activity, respiratory activity and membrane potential seem to suggest that the primary mechanism of action of coriander oil is membrane damage, which leads to cell death. The results obtained herein further encourage the use of coriander oil in antibacterial formulations due to the fact that coriander oil effectively kills pathogenic bacteria related to foodborne diseases and hospital infections.

  14. Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.

    Directory of Open Access Journals (Sweden)

    Chaves-Pozo Elena

    2004-01-01

    Full Text Available The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1&bgr;. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification.

  15. 16S rRNA in situ Hybridization Followed by Flow Cytometry for Rapid Identification of Acetic Acid Bacteria Involved in Submerged Industrial Vinegar Production

    Directory of Open Access Journals (Sweden)

    Luka Lipoglavšek

    2016-01-01

    Full Text Available Acetic acid bacteria are involved in many biotechnological processes such as vitamin C, gluconic acid, miglitol or acetic acid production, and others. For a technologist trying to control the industrial process, the ability to follow the microbiological development of the process is thus of importance. During the past few years hybridization in a combination with flow cytometry has often been used for this purpose. Since vinegar is a liquid, it is an ideal matrix for flow cytometry analysis. In this work we have constructed a specific probe for highly acetic acid-resistant species of the acetic acid bacteria and a protocol for in situ hybridization, which in combination with flow cytometry enables direct monitoring of bacteria producing vinegar with >10 % of acetic acid. The approach was successfully applied for monitoring microbiota during industrial vinegar production.

  16. Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes

    DEFF Research Database (Denmark)

    Jensen, Uffe Birk; Owens, David; Pedersen, Søren

    2010-01-01

    Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde in the pre......Zinc salt-based fixation (ZBF) has proved advantageous in histochemical analyses conducted on intact tissues but has not been exploited in flow cytometry procedures that focus on quantitative analysis of individual cells. Here, we show that ZBF performs equally well to paraformaldehyde...... allowing subsequent quantitative PCR analysis or labeling for incorporation of the thymidine analog EdU following surface and intracellular epitope staining. Finally, ZBF treatment allows for long-term storage of labeled cells with little change in these parameters. Thus, we present a protocol for zinc...... salt fixation of cells that allows for the simultaneous analysis of DNA and intracellular and cell surface proteins by flow cytometry....

  17. Real time kinetic flow cytometry measurements of cellular parameter changes evoked by nanosecond pulsed electric field.

    Science.gov (United States)

    Orbán, Csaba; Pérez-García, Esther; Bajnok, Anna; McBean, Gethin; Toldi, Gergely; Blanco-Fernandez, Alfonso

    2016-05-01

    Nanosecond pulsed electric field (nsPEF) is a novel method to increase cell proliferation rate. The phenomenon is based on the microporation of cellular organelles and membranes. However, we have limited information on the effects of nsPEF on cell physiology. Several studies have attempted to describe the effects of this process, however no real time measurements have been conducted to date. In this study we designed a model system which allows the measurement of cellular processes before, during and after nsPEF treatment in real time. The system employs a Vabrema Mitoplicator(TM) nsPEF field generating instrument connected to a BD Accuri C6 cytometer with a silicon tube led through a peristaltic pump. This model system was applied to observe the effects of nsPEF in mammalian C6 glioblastoma (C6 glioma) and HEK-293 cell lines. Viability (using DRAQ7 dye), intracellular calcium levels (using Fluo-4 dye) and scatter characteristics were measured in a kinetic manner. Data were analyzed using the FACSKin software. The viability and morphology of the investigated cells was not altered upon nsPEF treatment. The response of HEK-293 cells to ionomycin as positive control was significantly lower in the nsPEF treated samples compared to non-treated cells. This difference was not observed in C6 cells. FSC and SSC values were not altered significantly by the nsPEF treatment. Our results indicate that this model system is capable of reliably investigating the effects of nsPEF on