Report of the results of the International Clinical Cytometry Society and American Society for Clinical Pathology workload survey of clinical flow cytometry laboratories.

Science.gov (United States)

Wolniak, Kristy; Goolsby, Charles; Choi, Sarah; Ali, Asma; Serdy, Nina; Stetler-Stevenson, Maryalice

2017-11-01

Thorough review of current workload, staffing, and testing practices in clinical laboratories allows for optimization of laboratory efficiency and quality. This information is largely missing with regard to clinical flow cytometry laboratories. The purpose of this survey is to provide comprehensive, current, and accurate data on testing practices and laboratory staffing in clinical laboratories performing flow cytometric studies. Survey data was collected from flow cytometry laboratories through the ASCP website. Data was collected on the workload during a 1-year time period of full-time and part-time technical and professional (M.D./D.O./Ph.D. or equivalent) flow cytometry employees. Workload was examined as number of specimens and tubes per full time equivalent (FTE) technical and professional staff. Test complexity, test result interpretation, and reporting practices were also evaluated. There were 205 respondent laboratories affiliated predominantly with academic and health system institutions. Overall, 1,132 FTE employees were reported with 29% professional FTE employees and 71% technical. Fifty-one percent of the testing performed was considered high complexity and 49% was low complexity. The average number of tubes per FTE technologist was 1,194 per year and the average number of specimens per FTE professional was 1,659 per year. The flow cytometry reports were predominantly written by pathologists (57%) and were typically written as a separate report (58%). This survey evaluates the overall status of the current practice of clinical flow cytometry and provides a comprehensive dataset as a framework to help laboratory departments, directors, and managers make appropriate, cost-effective staffing decisions. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

Analysis of repetitive DNA in chromosomes by flow cytometry

NARCIS (Netherlands)

Brind'Amour, Julie; Lansdorp, Peter M.

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

Immune response to mycobacterial infection: lessons from flow cytometry.

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Rovina, Nikoletta; Panagiotou, Marios; Pontikis, Konstantinos; Kyriakopoulou, Magdalini; Koulouris, Nikolaos G; Koutsoukou, Antonia

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb) at certain stages of infection as revealed by flow cytometry to date.

Immune Response to Mycobacterial Infection: Lessons from Flow Cytometry

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Nikoletta Rovina

2013-01-01

Full Text Available Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active and latent TB: it summarizes diagnostic biomarkers distinguishing the two states of infection and also features of the distinct immune response against Mycobacterium tuberculosis (Mtb at certain stages of infection as revealed by flow cytometry to date.

A method for the interpretation of flow cytometry data using genetic algorithms

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Cesar Angeletti

2018-01-01

Full Text Available Background: Flow cytometry analysis is the method of choice for the differential diagnosis of hematologic disorders. It is typically performed by a trained hematopathologist through visual examination of bidimensional plots, making the analysis time-consuming and sometimes too subjective. Here, a pilot study applying genetic algorithms to flow cytometry data from normal and acute myeloid leukemia subjects is described. Subjects and Methods: Initially, Flow Cytometry Standard files from 316 normal and 43 acute myeloid leukemia subjects were transformed into multidimensional FITS image metafiles. Training was performed through introduction of FITS metafiles from 4 normal and 4 acute myeloid leukemia in the artificial intelligence system. Results: Two mathematical algorithms termed 018330 and 025886 were generated. When tested against a cohort of 312 normal and 39 acute myeloid leukemia subjects, both algorithms combined showed high discriminatory power with a receiver operating characteristic (ROC curve of 0.912. Conclusions: The present results suggest that machine learning systems hold a great promise in the interpretation of hematological flow cytometry data.

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

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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.

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...... for enumerating live parasites in bioassays was developed. The technique was applied to estimate the specific growth inhibition index (SGI) in the antibody-dependent cellular inhibition (ADCI) assay and compared to parasite quantification by microscopy and mitotracker red staining. The Bland-Altman analysis...

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

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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

Wide-field fluorescent microscopy and fluorescent imaging flow cytometry on a cell-phone.

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Zhu, Hongying; Ozcan, Aydogan

2013-04-11

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. - 10 μm over a very large field-of-view of - 81 mm(2). This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

A CLIPS expert system for clinical flow cytometry data analysis

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Salzman, G. C.; Duque, R. E.; Braylan, R. C.; Stewart, C. C.

1990-01-01

An expert system is being developed using CLIPS to assist clinicians in the analysis of multivariate flow cytometry data from cancer patients. Cluster analysis is used to find subpopulations representing various cell types in multiple datasets each consisting of four to five measurements on each of 5000 cells. CLIPS facts are derived from results of the clustering. CLIPS rules are based on the expertise of Drs. Stewart, Duque, and Braylan. The rules incorporate certainty factors based on case histories.

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

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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.

Misty Mountain clustering: application to fast unsupervised flow cytometry gating

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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

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

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Matamoros-Volante, Arturo; Moreno-Irusta, Ayelen; Torres-Rodriguez, Paulina; Giojalas, Laura; Gervasi, María G; Visconti, Pablo E; Treviño, Claudia L

2018-02-01

Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the

Hyperexpansion of wheat chromosomes sorted by flow cytometry

Czech Academy of Sciences Publication Activity Database

Endo, Takashi R.; Kubaláková, Marie; Vrána, Jan; Doležel, Jaroslav

2014-01-01

Roč. 89, č. 4 (2014), s. 181-185 ISSN 1341-7568 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : flow cytometry * flow sorting * chromosome Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.930, year: 2014 http://gateway.isiknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=MEDLINE&DestLinkType=FullRecord&UT=25747042

[Flow cytometry in datecting lymph node micrometastasis in colorectal cancer].

Science.gov (United States)

Sun, Q; Ding, Y; Zhang, J

2001-01-25

To study the methodology and significance of flow cytometry in detecting lymph node micrometastasis of colorectal cancer. One hundred sixty-two cellular suspensions were prepared with lymph nodes which were resected radically on 25 patients with colorectal cancer and in which no cancer cells were found by HE staining. Different concentrations of cultured Lovo colorectal cancer cells were added into the celular suspension prepared from lymph node tissue of persons without colorectal cancer in order to prepare a control model. Dual staining with CK/FTTC and PI was made to the sedimetns from those 2 kinds of suspension. Flow cytometry was used to detect cancer cells. An ideal correlation was obtained between the detection value and the theoretical value of cancer cells in the specimen suspensions and control models (r = 0.097 6) with a sensitivity rate of 10/10(5). Cancer cells were detected from 7 out of the 25 patients and 30 of the 162 cellular suspensions. The detection rate was correlated with the size and infiltrating depth of the cancer. Flow cytometry is a reliable, rapid, and quantitative method for detecting lymph node micrometastasis in colorectal cancer.

Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes

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Pipy Bernard

2010-02-01

Full Text Available Abstract Background The activity of promising anti-malarial drugs against Plasmodium gametocytes is hard to evaluate even in vitro. This is because visual examination of stained smears, which is commonly used, is not totally convenient. In the current study, flow cytometry has been used to study the effect of established anti-malarial drugs against sexual stages obtained from W2 strain of Plasmodium falciparum. Gametocytes were treated for 48 h with different drug concentrations and the gametocytaemia was then determined by flow cytometry and compared with visual estimation by microscopy. Results and conclusions Initially gametocytaemia was evaluated either using light microscopy or flow cytometry. A direct correlation (r2 = 0.9986 was obtained. Two distinct peaks were observed on cytometry histograms and were attributed to gametocyte populations. The activities of established anti-malarial compounds were then measured by flow cytometry and the results were equivalent to those obtained using light microscopy. Primaquine and artemisinin had IC50 of 17.6 μM and 1.0 μM, respectively. Gametocyte sex was apparently distinguishable by flow cytometry as evaluated after induction of exflagellation by xanthurenic acid. These data form the basis of further studies for developing new methods in drug discovery to decrease malaria transmission.

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

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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.

Surface profiling of normally responding and nonreleasing basophils by flow cytometry

DEFF Research Database (Denmark)

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

a maximum release blood mononuclear cells were purified by density centrifugation and using flow cytometry, 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......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 (

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.

Optimizing transformations for automated, high throughput analysis of flow cytometry data.

Science.gov (United States)

Finak, Greg; Perez, Juan-Manuel; Weng, Andrew; Gottardo, Raphael

2010-11-04

In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. We compare the performance of parameter-optimized and default-parameter (in flowCore) data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter-optimized transformations improve visualization, reduce

Optimizing transformations for automated, high throughput analysis of flow cytometry data

Directory of Open Access Journals (Sweden)

Weng Andrew

2010-11-01

Full Text Available Abstract Background In a high throughput setting, effective flow cytometry data analysis depends heavily on proper data preprocessing. While usual preprocessing steps of quality assessment, outlier removal, normalization, and gating have received considerable scrutiny from the community, the influence of data transformation on the output of high throughput analysis has been largely overlooked. Flow cytometry measurements can vary over several orders of magnitude, cell populations can have variances that depend on their mean fluorescence intensities, and may exhibit heavily-skewed distributions. Consequently, the choice of data transformation can influence the output of automated gating. An appropriate data transformation aids in data visualization and gating of cell populations across the range of data. Experience shows that the choice of transformation is data specific. Our goal here is to compare the performance of different transformations applied to flow cytometry data in the context of automated gating in a high throughput, fully automated setting. We examine the most common transformations used in flow cytometry, including the generalized hyperbolic arcsine, biexponential, linlog, and generalized Box-Cox, all within the BioConductor flowCore framework that is widely used in high throughput, automated flow cytometry data analysis. All of these transformations have adjustable parameters whose effects upon the data are non-intuitive for most users. By making some modelling assumptions about the transformed data, we develop maximum likelihood criteria to optimize parameter choice for these different transformations. Results We compare the performance of parameter-optimized and default-parameter (in flowCore data transformations on real and simulated data by measuring the variation in the locations of cell populations across samples, discovered via automated gating in both the scatter and fluorescence channels. We find that parameter

Stochastic Measurement Models for Quantifying Lymphocyte Responses Using Flow Cytometry

Science.gov (United States)

Kan, Andrey; Pavlyshyn, Damian; Markham, John F.; Dowling, Mark R.; Heinzel, Susanne; Zhou, Jie H. S.; Marchingo, Julia M.; Hodgkin, Philip D.

2016-01-01

Adaptive immune responses are complex dynamic processes whereby B and T cells undergo division and differentiation triggered by pathogenic stimuli. Deregulation of the response can lead to severe consequences for the host organism ranging from immune deficiencies to autoimmunity. Tracking cell division and differentiation by flow cytometry using fluorescent probes is a major method for measuring progression of lymphocyte responses, both in vitro and in vivo. In turn, mathematical modeling of cell numbers derived from such measurements has led to significant biological discoveries, and plays an increasingly important role in lymphocyte research. Fitting an appropriate parameterized model to such data is the goal of these studies but significant challenges are presented by the variability in measurements. This variation results from the sum of experimental noise and intrinsic probabilistic differences in cells and is difficult to characterize analytically. Current model fitting methods adopt different simplifying assumptions to describe the distribution of such measurements and these assumptions have not been tested directly. To help inform the choice and application of appropriate methods of model fitting to such data we studied the errors associated with flow cytometry measurements from a wide variety of experiments. We found that the mean and variance of the noise were related by a power law with an exponent between 1.3 and 1.8 for different datasets. This violated the assumptions inherent to commonly used least squares, linear variance scaling and log-transformation based methods. As a result of these findings we propose a new measurement model that we justify both theoretically, from the maximum entropy standpoint, and empirically using collected data. Our evaluation suggests that the new model can be reliably used for model fitting across a variety of conditions. Our work provides a foundation for modeling measurements in flow cytometry experiments thus

Antimicrobial activity and cytotoxicity of piperazinium- and guanidinium-based ionic liquids

Energy Technology Data Exchange (ETDEWEB)

Yu, Jing; Zhang, Shanshan; Dai, Yitong; Lu, Xiaoxing; Lei, Qunfang; Fang, Wenjun, E-mail: qflei@zju.edu.cn

2016-04-15

Highlights: • Twelve piperazinium- and guanidinium-based ionic liquids were synthesized and characterized. • Antimicrobial activities of the ionic liquids against E. coli and S. aureus were investigated. • Cytotoxicity on the rat C6 glioma cells (C6) and human embryonic kidney cells (HEK-293) were evaluated. • The ionic liquids with the [BF{sub 4}]{sup −} anion and with benzene ring on cation exhibit relatively high toxicity. - Abstract: Twelve piperazinium- and guanidinium-based ionic liquids (ILs) were synthesized, and characterized by {sup 1}H nuclear magnetic resonance (NMR), thermal gravimetric analyzer (TGA) and differential scanning calorimetry (DSC). The antimicrobial activity and cytotoxicity have been investigated to provide the information whether the newly synthesized ILs are toxic or not. The antimicrobial effects of these ILs on gram negative and gram positive bacteria are evaluated on the basis of the minimum inhibitory concentration (MIC) measurements. The membrane damages of bacteria in the presence of ILs are observed by scanning electron microscopy (SEM). The cytotoxicity data of the ILs on HEK-293 and C6 cells are obtained by MTT cell viability assay. The disruption of cell cycle is analyzed by the flow cytometry. The results show that most of the ILs exhibit low toxicity, and the ILs with tetrafluoroborate anion and with benzene ring on cation are the species with relatively high toxicity among the studied ILs. The fundamental data and results can provide some useful information for the further studies and applications of the ILs.

Antimicrobial activity and cytotoxicity of piperazinium- and guanidinium-based ionic liquids

International Nuclear Information System (INIS)

Yu, Jing; Zhang, Shanshan; Dai, Yitong; Lu, Xiaoxing; Lei, Qunfang; Fang, Wenjun

2016-01-01

Highlights: • Twelve piperazinium- and guanidinium-based ionic liquids were synthesized and characterized. • Antimicrobial activities of the ionic liquids against E. coli and S. aureus were investigated. • Cytotoxicity on the rat C6 glioma cells (C6) and human embryonic kidney cells (HEK-293) were evaluated. • The ionic liquids with the [BF_4]"− anion and with benzene ring on cation exhibit relatively high toxicity. - Abstract: Twelve piperazinium- and guanidinium-based ionic liquids (ILs) were synthesized, and characterized by "1H nuclear magnetic resonance (NMR), thermal gravimetric analyzer (TGA) and differential scanning calorimetry (DSC). The antimicrobial activity and cytotoxicity have been investigated to provide the information whether the newly synthesized ILs are toxic or not. The antimicrobial effects of these ILs on gram negative and gram positive bacteria are evaluated on the basis of the minimum inhibitory concentration (MIC) measurements. The membrane damages of bacteria in the presence of ILs are observed by scanning electron microscopy (SEM). The cytotoxicity data of the ILs on HEK-293 and C6 cells are obtained by MTT cell viability assay. The disruption of cell cycle is analyzed by the flow cytometry. The results show that most of the ILs exhibit low toxicity, and the ILs with tetrafluoroborate anion and with benzene ring on cation are the species with relatively high toxicity among the studied ILs. The fundamental data and results can provide some useful information for the further studies and applications of the ILs.

Hyperspectral cytometry.

Science.gov (United States)

Grégori, Gérald; Rajwa, Bartek; Patsekin, Valery; Jones, James; Furuki, Motohiro; Yamamoto, Masanobu; Paul Robinson, J

2014-01-01

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

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.

Real-time Image Processing for Microscopy-based Label-free Imaging Flow Cytometry in a Microfluidic Chip.

Science.gov (United States)

Heo, Young Jin; Lee, Donghyeon; Kang, Junsu; Lee, Keondo; Chung, Wan Kyun

2017-09-14

Imaging flow cytometry (IFC) is an emerging technology that acquires single-cell images at high-throughput for analysis of a cell population. Rich information that comes from high sensitivity and spatial resolution of a single-cell microscopic image is beneficial for single-cell analysis in various biological applications. In this paper, we present a fast image-processing pipeline (R-MOD: Real-time Moving Object Detector) based on deep learning for high-throughput microscopy-based label-free IFC in a microfluidic chip. The R-MOD pipeline acquires all single-cell images of cells in flow, and identifies the acquired images as a real-time process with minimum hardware that consists of a microscope and a high-speed camera. Experiments show that R-MOD has the fast and reliable accuracy (500 fps and 93.3% mAP), and is expected to be used as a powerful tool for biomedical and clinical applications.

Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

Science.gov (United States)

Käser, T; Pasternak, J A; Hamonic, G; Rieder, M; Lai, K; Delgado-Ortega, M; Gerdts, V; Meurens, F

2016-05-01

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Lipid nanoparticles assessment by flow cytometry.

Science.gov (United States)

Bryła, Anna; Juzwa, Wojciech; Weiss, Marek; Lewandowicz, Grażyna

2017-03-30

Liposomes are promising carriers for drugs and bioactive compounds. Size and structure are their crucial parameters. Thus, it is essential to assess individual vesicles as prepared. Currently available techniques fail to measure liposome's size and structure simultaneously, with a high throughput. To solve this problem, we have developed a novel, flow cytometric method quantifying liposomes. Firstly, the following fluorescent staining combinations were tested: DiD/TO, Rh123/DiD, Syto9/DiD. Further, chosen fluorochromes were used to compare three populations of vesicles: raw (R), obtained by thin film hydration and extruded ones (populations E10 and E21). Dynamic light scattering (DLS) was used for determination of average diameter and size distribution of nanocarriers. Structural differences between the raw and the extruded liposomes, as well as additional information concerning vesicles size were acquired employing atomic force microscopy (AFM). DLS analysis indicated that, three distinct populations of vesicles were obtained. Liposomes were characterized by mean diameter of 323nm, 220nm and 170nm for population R, E10 and E21 respectively. All the populations were stable and revealed zeta potential of -29mV. AFM confirmed that raw and extruded liposomes were differed in structure. DiD/TO was the optimal fluorochrome combination that enabled to resolve distinctly the sub-populations of liposomes. Results obtained by flow cytometry were in a good agreement with those from DLS and AFM. It was proved that, flow cytometry, when proper fluorescent dyes are used, is an adequate method for liposomes assessment. The proposed method enables fast and reliable analysis of liposomes in their native environment. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinical cytometry and progress in HLA antibody detection.

Science.gov (United States)

Bray, Robert A; Tarsitani, Christine; Gebel, Howard M; Lee, Jar-How

2011-01-01

For most solid organ and selected stem cell transplants, antibodies against mismatched HLA antigens can lead to early and late graft failure. In recognition of the clinical significance of these antibodies, HLA antibody identification is one of the most critical functions of histocompatibility laboratories. Early methods employed cumbersome and insensitive complement-dependent cytotoxicity assays with a visual read-out. A little over 20 years ago flow cytometry entered the realm of antibody detection with the introduction of the flow cytometric crossmatch. Cytometry's increased sensitivity and objectivity quickly earned it popularity as a preferred crossmatch method especially for sensitized recipients. Although a sensitive method, the flow crossmatch was criticized as being "too sensitive" as false positive reactions were a know drawback. In part, the shortcomings of the flow crossmatch were due to the lack of corresponding sensitive and specific HLA antibody screening assays. However, in the mid 1990s, solid phase assays, capable of utilizing standard flow cytometers, were developed. These assays used microparticles coated with purified HLA molecules. Hence, the era of solid-phase, microparticle technology for HLA antibody detection was born permitting the sensitive and specific detection of HLA antibody. It was now possible to provide better correlation between HLA antibody detection and the flow cytometric crossmatch. This flow-based technology was soon followed by adaptation to the Luminex platform permitting a mutltiplexed approach for the identification and characterization of HLA antibodies. It is hoped that these technologies will ultimately lead to the identification of parameters that best correlate with and/or predict transplant outcomes. Copyright © 2011 Elsevier Inc. All rights reserved.

Methodology and application of flow cytometry for investigation of human malaria parasites.

Science.gov (United States)

Grimberg, Brian T

2011-03-31

Historically, examinations of the inhibition of malaria parasite growth/invasion, whether using drugs or antibodies, have relied on the use of microscopy or radioactive hypoxanthine uptake. These are considered gold standards for measuring the effectiveness of antimalarial treatments, however, these methods have well known shortcomings. With the advent of flow cytometry coupled with the use of fluorescent DNA stains allowed for increased speed, reproducibility, and qualitative estimates of the effectiveness of antibodies and drugs to limit malaria parasite growth which addresses the challenges of traditional techniques. Because materials and machines available to research facilities are so varied, different methods have been developed to investigate malaria parasites by flow cytometry. This review is intended to serve as a reference guide for advanced users and importantly, as a primer for new users, to support expanded use and improvements to malaria flow cytometry, particularly in endemic countries. Copyright © 2011 Elsevier B.V. All rights reserved.

flowPeaks: a fast unsupervised clustering for flow cytometry data via K-means and density peak finding.

Science.gov (United States)

Ge, Yongchao; Sealfon, Stuart C

2012-08-01

For flow cytometry data, there are two common approaches to the unsupervised clustering problem: one is based on the finite mixture model and the other on spatial exploration of the histograms. The former is computationally slow and has difficulty to identify clusters of irregular shapes. The latter approach cannot be applied directly to high-dimensional data as the computational time and memory become unmanageable and the estimated histogram is unreliable. An algorithm without these two problems would be very useful. In this article, we combine ideas from the finite mixture model and histogram spatial exploration. This new algorithm, which we call flowPeaks, can be applied directly to high-dimensional data and identify irregular shape clusters. The algorithm first uses K-means algorithm with a large K to partition the cell population into many small clusters. These partitioned data allow the generation of a smoothed density function using the finite mixture model. All local peaks are exhaustively searched by exploring the density function and the cells are clustered by the associated local peak. The algorithm flowPeaks is automatic, fast and reliable and robust to cluster shape and outliers. This algorithm has been applied to flow cytometry data and it has been compared with state of the art algorithms, including Misty Mountain, FLOCK, flowMeans, flowMerge and FLAME. The R package flowPeaks is available at https://github.com/yongchao/flowPeaks. yongchao.ge@mssm.edu Supplementary data are available at Bioinformatics online.

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

Cytometry metadata in XML

Science.gov (United States)

Leif, Robert C.; Leif, Stephanie H.

2016-04-01

Introduction: The International Society for Advancement of Cytometry (ISAC) has created a standard for the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt 1.0). CytometryML will serve as a common metadata standard for flow and image cytometry (digital microscopy). Methods: The MIFlowCyt data-types were created, as is the rest of CytometryML, in the XML Schema Definition Language (XSD1.1). The datatypes are primarily based on the Flow Cytometry and the Digital Imaging and Communication (DICOM) standards. A small section of the code was formatted with standard HTML formatting elements (p, h1, h2, etc.). Results:1) The part of MIFlowCyt that describes the Experimental Overview including the specimen and substantial parts of several other major elements has been implemented as CytometryML XML schemas (www.cytometryml.org). 2) The feasibility of using MIFlowCyt to provide the combination of an overview, table of contents, and/or an index of a scientific paper or a report has been demonstrated. Previously, a sample electronic publication, EPUB, was created that could contain both MIFlowCyt metadata as well as the binary data. Conclusions: The use of CytometryML technology together with XHTML5 and CSS permits the metadata to be directly formatted and together with the binary data to be stored in an EPUB container. This will facilitate: formatting, data- mining, presentation, data verification, and inclusion in structured research, clinical, and regulatory documents, as well as demonstrate a publication's adherence to the MIFlowCyt standard, promote interoperability and should also result in the textual and numeric data being published using web technology without any change in composition.

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.

CytometryML: a markup language for analytical cytology

Science.gov (United States)

Leif, Robert C.; Leif, Stephanie H.; Leif, Suzanne B.

2003-06-01

Cytometry Markup Language, CytometryML, is a proposed new analytical cytology data standard. CytometryML is a set of XML schemas for encoding both flow cytometry and digital microscopy text based data types. CytometryML schemas reference both DICOM (Digital Imaging and Communications in Medicine) codes and FCS keywords. These schemas provide representations for the keywords in FCS 3.0 and will soon include DICOM microscopic image data. Flow Cytometry Standard (FCS) list-mode has been mapped to the DICOM Waveform Information Object. A preliminary version of a list mode binary data type, which does not presently exist in DICOM, has been designed. This binary type is required to enhance the storage and transmission of flow cytometry and digital microscopy data. Index files based on Waveform indices will be used to rapidly locate the cells present in individual subsets. DICOM has the advantage of employing standard file types, TIF and JPEG, for Digital Microscopy. Using an XML schema based representation means that standard commercial software packages such as Excel and MathCad can be used to analyze, display, and store analytical cytometry data. Furthermore, by providing one standard for both DICOM data and analytical cytology data, it eliminates the need to create and maintain special purpose interfaces for analytical cytology data thereby integrating the data into the larger DICOM and other clinical communities. A draft version of CytometryML is available at www.newportinstruments.com.

Rapid detection of aneuploidy in Musa using flow cytometry

Czech Academy of Sciences Publication Activity Database

Roux, N.; Toloza, A.; Radecki, Z.; Zapata-Arias, F. J.; Doležel, Jaroslav

2003-01-01

Roč. 21, - (2003), s. 483-490 ISSN 0721-7714 R&D Projects: GA AV ČR IAA6038204 Institutional research plan: CEZ:AV0Z5038910 Keywords : banana * flow cytometry * nuclear DNA content Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.423, year: 2003

High resolution light-sheet based high-throughput imaging cytometry system enables visualization of intra-cellular organelles

Science.gov (United States)

Regmi, Raju; Mohan, Kavya; Mondal, Partha Pratim

2014-09-01

Visualization of intracellular organelles is achieved using a newly developed high throughput imaging cytometry system. This system interrogates the microfluidic channel using a sheet of light rather than the existing point-based scanning techniques. The advantages of the developed system are many, including, single-shot scanning of specimens flowing through the microfluidic channel at flow rate ranging from micro- to nano- lit./min. Moreover, this opens-up in-vivo imaging of sub-cellular structures and simultaneous cell counting in an imaging cytometry system. We recorded a maximum count of 2400 cells/min at a flow-rate of 700 nl/min, and simultaneous visualization of fluorescently-labeled mitochondrial network in HeLa cells during flow. The developed imaging cytometry system may find immediate application in biotechnology, fluorescence microscopy and nano-medicine.

Rapid detection of microbial contamination in grape juice by flow cytometry

Directory of Open Access Journals (Sweden)

Marielle Bouix

1999-03-01

Full Text Available This study presents an application of flow cytometry to evaluate rapidly the viable micro-organisms in grape juice. In this method, viable cells are firstly specitically labelled with a fluorescent reagent. The sample is then injected into the flow cytometer where the labelled micro-organisms are individually illuminated by a laser beam. The emission of fluorescence is measured. The system counts the number of fluorescent events and prints out a histogram of the fluorescence intensity which is characteristic of the micro-organism being analysed. In laboratory conditions, preliminary trials have been undertaken with an artificially inoculated grape juice with pure yeast and bacteria cultures. This method succeeded in counting simultaneously yeasts and bacteria within 15 minutes, with a high degree of sensitivity, 5.103 yeasts perml and 5.104 bacteria per ml. This technique can also be applied to the detection of mould contamination and the test has been done with Botrytis spores. The method makes direct cell counts possible and is capable of analysing 30 samples per hour. It can be automatised and easily used in industrial laboratory. During the last harvest, more than a thousand of must samples were controled using this technique. The results let to determine the yeast contamination level of a grape juice tank even before unloading. The results obtained by flow cytometry were compared to the plate count reference method. The correlation between cytometry and count by plate culture was 99 p. cent for the threshold of 5.1 04 yeasts/ml which seemed to point out a high contamination. By using this flow cytometry method during the harvest period, the results were supplied in real time. This allowed a rapid selection of the musts, depending upon the scale of their contamination and improved the quality of the wine by corrective actions.

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...... 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...... with those obtained by manual gating (r between 0.79 and 0.99) and outperformed other model-driven gating methods. Bland-Altman plots confirmed the agreement of manual gating and OSA derived results. A-1.33 fold increase (p=0.003) in the number of positive cells after vaccination in a subgroup of preschool...

Identification of contact and respiratory sensitizers using flow cytometry

International Nuclear Information System (INIS)

Goutet, Michele; Pepin, Elsa; Langonne, Isabelle; Huguet, Nelly; Ban, Masarin

2005-01-01

Identification of the chemicals responsible for respiratory and contact allergies in the industrial area is an important occupational safety issue. This study was conducted in mice to determine whether flow cytometry is an appropriate method to analyze and differentiate the specific immune responses to the respiratory sensitizer trimellitic anhydride (TMA) and to the contact sensitizer dinitrochlorobenzene (DNCB) used at concentrations with comparable immunogenic potential. Mice were exposed twice on the flanks (days 0, 5) to 10% TMA or 1% DNCB and challenged three times on the ears (days 10, 11, 12) with 2.5% TMA or 0.25% DNCB. Flow cytometry analyses were conducted on draining lymph node cells harvested on days 13 and 18. Comparing TMA and DNCB immune responses on day 13, we found obvious differences that persisted for most of them on day 18. An increased proportion of IgE+ cells correlated to total serum IgE level and an enhancement of MHC II molecule expression were observed in the lymph node B lymphocytes from TMA-treated mice. The percentage of IL-4-producing CD4+ lymphocytes and the IL-4 receptor expression were clearly higher following TMA exposure. In contrast, higher proportions of IL-2-producing cells were detected in CD4+ and CD8+ cells from DNCB-treated mice. Both chemicals induced a significant increase in the percentage of IFN-γ-producing cells among CD8+ lymphocytes but to a greater proportion following TMA treatment. In conclusion, this study encourages the use of flow cytometry to discriminate between contact and respiratory sensitizers by identifying divergent expression of immune response parameters

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. © 2014 International Society for Advancement of Cytometry.

Measurement and Characterization of Apoptosis by Flow Cytometry.

Science.gov (United States)

Telford, William; Tamul, Karen; Bradford, Jolene

2016-07-01

Apoptosis is an important mechanism in cell biology, playing a critical regulatory role in virtually every organ system. It has been particularly well characterized in the immune system, with roles ranging from immature immune cell development and selection to down-regulation of the mature immune response. Apoptosis is also the primary mechanism of action of anti-cancer drugs. Flow cytometry has been the method of choice for analyzing apoptosis in suspension cells for more than 25 years. Numerous assays have been devised to measure both the earliest and latest steps in the apoptotic process, from the earliest signal-transduction events to the late morphological changes in cell shape and granularity, proteolysis, and chromatin condensation. These assays are particularly powerful when combined into multicolor assays determining several apoptotic characteristics simultaneously. The multiparametric nature of flow cytometry makes this technology particularly suited to measuring apoptosis. In this unit, we will describe the four main techniques for analyzing caspase activity in apoptotic cells, combined with annexin V and cell permeability analysis. These relatively simple multiparametric assays are powerful techniques for assessing cell death. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Simultaneous ultrasound and photoacoustics based flow cytometry

Science.gov (United States)

Gnyawali, Vaskar; Strohm, Eric M.; Tsai, Scott S. H.; Kolios, Michael C.

2018-04-01

We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

Science.gov (United States)

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

A general method for bead-enhanced quantitation by flow cytometry

Science.gov (United States)

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

Determination of total bacterial count in raw milk by flow cytometry

Directory of Open Access Journals (Sweden)

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.

Flow Cytometry Method as a Diagnostic Tool for Pleural Fluid Involvement in a Patient with Multiple Myeloma

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Muzaffer Keklik

2012-10-01

Full Text Available Multiple myeloma is a malignant proliferation of plasma cells that mainly affects bone marrow. Pleural effusions secondary to pleural myelomatous involvement have rarely been reported in the literature. As it is rarely detected, we aimed to report a case in which pleural effusion of a multiple myeloma was confirmed as true myelomatous involvement by flow cytometry method. A 52-years old man presented to our clinic with chest and back pain lasting for 3 months. On the chest radiography, pleural fluid was detected in left hemithorax. Pleural fluid flow cytometry was performed. In the flow cytometry, CD56, CD38 and CD138 found to be positive, while CD19 was negative. True myelomatous pleural effusions are very uncommon, with fewer than 100 cases reported worldwide. Flow cytometry is a potentially useful diagnostic tool for clinical practice. We presented our case; as it has been rarely reported, although flow cytometer is a simple method for detection of pleural fluid involvement in multiple myeloma.

Interlaboratory assessment of mitotic index by flow cytometry confirms superior reproducibility relative to microscopic scoring.

Science.gov (United States)

Roberts, D J; Spellman, R A; Sanok, K; Chen, H; Chan, M; Yurt, P; Thakur, A K; DeVito, G L; Murli, H; Stankowski, L F

2012-05-01

A flow cytometric procedure for determining mitotic index (MI) as part of the metaphase chromosome aberrations assay, developed and utilized routinely at Pfizer as part of their standard assay design, has been adopted successfully by Covance laboratories. This method, using antibodies against phosphorylated histone tails (H3PS10) and nucleic acid stain, has been evaluated by the two independent test sites and compared to manual scoring. Primary human lymphocytes were treated with cyclophosphamide, mitomycin C, benzo(a)pyrene, and etoposide at concentrations inducing dose-dependent cytotoxicity. Deming regression analysis indicates that the results generated via flow cytometry (FCM) were more consistent between sites than those generated via microscopy. Further analysis using the Bland-Altman modification of the Tukey mean difference method supports this finding, as the standard deviations (SDs) of differences in MI generated by FCM were less than half of those generated manually. Decreases in scoring variability owing to the objective nature of FCM, and the greater number of cells analyzed, make FCM a superior method for MI determination. In addition, the FCM method has proven to be transferable and easily integrated into standard genetic toxicology laboratory operations. Copyright © 2012 Wiley Periodicals, Inc.

Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

Science.gov (United States)

Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

2015-01-01

During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

Accuracy of Automated Flow Cytometry-Based Leukocyte Counts To Rule Out Urinary Tract Infection in Febrile Children: a Prospective Cross-Sectional Study

Science.gov (United States)

Duong, Hong Phuoc; Wissing, Karl Martin; Tram, Nathalie; Mascart, Georges; Lepage, Philippe

2016-01-01

Automated flow cytometry of urine remains an incompletely validated method to rule out urinary tract infection (UTI) in children. This cross-sectional analytical study was performed to compare the predictive values of flow cytometry and a dipstick test as initial diagnostic tests for UTI in febrile children and prospectively included 1,106 children (1,247 episodes). Urine culture was used as the gold standard test for diagnosing UTI. The performance of screening tests to diagnose UTI were established using receiver operating characteristic (ROC) analysis. Among these 1,247 febrile episodes, 221 UTIs were diagnosed (17.7% [95% confidence interval {CI}, 15.6 to 19.8%]). The area under the ROC curve for flow cytometry white blood cell (WBC) counts (0.99 [95% CI, 0.98 to 0.99]) was significantly superior to that for red blood cell (0.74 [95% CI, 0.70 to 0.78]) and bacterial counts (0.89 [95% CI, 0.87 to 0.92]) (P UTI in febrile children. PMID:27682127

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

Science.gov (United States)

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

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. © 2016 Society for Laboratory Automation and Screening.

Sample handling for kinetics and molecular assembly in flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Sklar, L.A. [Los Alamos National Lab., NM (United States). National Flow Cytometry Resource]|[Univ. of New Mexico, Albuquerque, NM (United States). School of Medicine; Seamer, L.C.; Kuckuck, F.; Prossnitz, E.; Edwards, B. [Univ. of New Mexico, Albuquerque, NM (United States). School of Medicine; Posner, G. [Northern Arizona Univ., Flagstaff, AZ (United States). Dept. of Chemistry

1998-07-01

Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. The authors have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, the authors used fast valves to overcome the disparity between mixing 100 {micro}ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 {micro}l/sec. Particles were detected within 100 msec after mixing, but turbulence was created which lasted for 1 sec after injection of the sample into the flow cytometer. They used optical criteria to discriminate particles which were out of alignment due to the turbulent flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system they were able to automate sample handling and delivery with intervals of a few seconds. The authors used a fluidic approach to defeat turbulence caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of turbulence was reduced to {approximately} 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

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

refractive index matching conditions and by cells labeled by gold nanoparticles. The optical schematics including phase contrast (OPCM) microscopy as a prospective modality for in vivo flow cytometry is also analyzed. The validation of the FDTD approach for the simulation of flow cytometry may open a new...

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.

Detection of palytoxin-like compounds by a flow cytometry-based immunoassay supported by functional and analytical methods.

Science.gov (United States)

Fraga, María; Vilariño, Natalia; Louzao, M Carmen; Fernández, Diego A; Poli, Mark; Botana, Luis M

2016-01-15

Palytoxin (PLTX) is a complex marine toxin produced by zoanthids (i.e. Palythoa), dinoflagellates (Ostreopsis) and cyanobacteria (Trichodesmium). PLTX outbreaks are usually associated with Indo-Pacific waters, however their recent repeated occurrence in Mediterranean-European Atlantic coasts demonstrate their current worldwide distribution. Human sickness and fatalities have been associated with toxic algal blooms and ingestion of seafood contaminated with PLTX-like molecules. These toxins represent a serious threat to human health. There is an immediate need to develop easy-to-use, rapid detection methods due to the lack of validated protocols for their detection and quantification. We have developed an immuno-detection method for PLTX-like molecules based on the use of microspheres coupled to flow-cytometry detection (Luminex 200™). The assay consisted of the competition between free PLTX-like compounds in solution and PLTX immobilized on the surface of microspheres for binding to a specific monoclonal anti-PLTX antibody. This method displays an IC50 of 1.83 ± 0.21 nM and a dynamic range of 0.47-6.54 nM for PLTX. An easy-to-perform extraction protocol, based on a mixture of methanol and acetate buffer, was applied to spiked mussel samples providing a recovery rate of 104 ± 8% and a range of detection from 374 ± 81 to 4430 ± 150 μg kg(-1) when assayed with this method. Extracts of Ostreopsis cf. siamensis and Palythoa tuberculosa were tested and yielded positive results for PLTX-like molecules. However, the data obtained for the coral sample suggested that this antibody did not detect 42-OH-PLTX efficiently. The same samples were further analyzed using a neuroblastoma cytotoxicity assay and UPLC-IT-TOF spectrometry, which also pointed to the presence of PLTX-like compounds. Therefore, this single detection method for PLTX provides a semi-quantitative tool useful for the screening of PLTX-like molecules in different matrixes. Copyright © 2015

Measuring T cell-mediated cytotoxicity using fluorogenic caspase substrates.

Science.gov (United States)

Chahroudi, A; Silvestri, G; Feinberg, M B

2003-10-01

Cytotoxic T lymphocytes (CTLs) play a major role in the immune response against viruses and other intracellular pathogens. In addition, CTLs are implicated in the control of tumor cells in certain settings. Accurate measures of CTL function are of critical importance to study the pathogenesis of infectious diseases and to evaluate the efficacy of new vaccines and immunotherapies. To this end, we have recently developed a flow cytometry-based CTL (FCC) assay that measures the CTL-induced caspase activation within target cells using cell permeable fluorogenic caspase substrates. This novel assay reliably detects, by flow cytometry or fluorescence/confocal microscopy, antigen-specific CTLs in a wide variety of human and murine systems, and is safer and more informative than the standard 51Cr-release assay. In addition, the flow cytometric CTL (FCC) assay provides an alternative method that is often more sensitive and physiologically informative when compared to previously described FCC assays, as it measures a biological indicator of apoptosis within the target cell. The FCC assay may thus represent a useful tool to further understand the molecular and cellular mechanisms that underlie CTL-mediated killing during tumorigenesis or following infection with viruses or other intracellular pathogens.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

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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.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

Science.gov (United States)

Alves, L P S; Almeida, A T; Cruz, L M; Pedrosa, F O; de Souza, E M; Chubatsu, L S; Müller-Santos, M; Valdameri, G

2017-01-16

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.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

Science.gov (United States)

Alves, L.P.S.; Almeida, A.T.; Cruz, L.M.; Pedrosa, F.O.; de Souza, E.M.; Chubatsu, L.S.; Müller-Santos, M.; Valdameri, G.

2017-01-01

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. PMID:28099582

Screening of carcinoma metastasis by flow cytometry: A study of 238 cases.

Science.gov (United States)

Acosta, Maria; Pereira, José; Arroz, Maria

2016-05-01

Malignant epithelial cells may be detected in different specimens, by immunophenotyping using flow cytometry (FCM). CD326 (epithelial-specific antigen, clone Ber-Ep4) was used to identify epithelial cells, CD45 to discriminate between leucocytes (positive for this antigen) and non-hematological cells (negative for this antigen), and CD33 to identify monocytes/macrophages. This combination is particularly useful in effusions to characterize large cells and distinguish between monocyte/macrophages (CD45+ CD33+ CD326-), mesothelial cells (CD45 ± (dim) CD33 - CD326-) and epithelial cells (CD45 - CD33 - CD326 +). We evaluated the efficiency of flow cytometry to detect malignant epithelial cells in 238 fresh samples, including effusions, lymph node biopsies, fine needle aspirates, bone marrow aspirates, cerebrospinal fluid, among others. These are specimens expected to lack epithelial cells. FCM results were then compared to the results of smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks, when available. Final diagnosis was the gold standard and a very good sensitivity (96.7%) and specificity (99.3%) were obtained. We concluded that the detection of CD326 positive cells using FCM is strongly indicative of the presence of carcinoma cells. © 2015 International Clinical Cytometry Society. © 2015 International Clinical Cytometry Society.

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

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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.

OpenCyto: an open source infrastructure for scalable, robust, reproducible, and automated, end-to-end flow cytometry data analysis.

Directory of Open Access Journals (Sweden)

Greg Finak

2014-08-01

Full Text Available Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

Science.gov (United States)

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Applications of flow cytometry to toxicological mycotoxin effects in cultured mammalian cells: a review.

Science.gov (United States)

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-06-01

This review gives an overview of flow cytometry applications to toxicological studies of several physiological target sites of mycotoxins on different mammalian cell lines. Mycotoxins are secondary metabolites of fungi that may be present in food, feed, air and water. The increasing presence of mycotoxins in crops, their wide distribution in the food chain, and their potential for toxicity demonstrate the need for further knowledge. Flow cytometry has become a valuable tool in mycotoxin studies in recent years for the rapid analysis of single cells in a mixture. In toxicology, the power of these methods lies in the possibility of determining a wide range of cell parameters, providing valuable information to elucidate cell growth and viability, metabolic activity, mitochondrial membrane potential and membrane integrity mechanisms. There are studies using flow cytometry technique on Alternaria, Aspergillus, Fusarium and Penicillium mycotoxins including information about cell type, assay conditions and functional parameters. Most of the studies collected in the literature are on deoxynivalenol and zearalenone mycotoxins. Cell cycle analysis and apoptosis are the processes more widely investigated. Copyright © 2013 Elsevier Ltd. All rights reserved.

Analysis of immunophenotype in acute myeloid leukemia by multiparameter flow cytometry

International Nuclear Information System (INIS)

Gao Yanqun; Jin Haijie; Yan Pei; Wang Feifei; Li Xiaohong; Gao Chunji

2005-01-01

To evaluate the immunophenotype of acute leukemia patients, the surface and cytoplasmic antigen expression in 162 cases of acute leukemia were analyzed by multiparameter flow cytometry and CD45/SSC gating. The results showed that CDl17 (94.9%), CD13 (88.5%) and CD33(70.5%) were mainly expressed in ANLL patients; cCD79a(100%), CD19(92.1%) were chiefly expressed in B-ALL patients, and in T-ALL patients, cCD3(100%) and CD2(83.3%) were expressed; For the expression of lymphoid differentiation antigen Ly+ANLL, CD7 (56.2%) and CD19(31.2%) were chiefly found, and for myeloid antigen My+ALL, CD13(88. 9%) and CD33 (27.8%) were detected. In conclusion, multiparameter flow cytometry and three-color direct immunofluorescence staining methods may be of important clinical significance in diagnosis, therapy and prognosis of acute leukemia. (authors)

Use of Multicolor Flow Cytometry for Isolation of Specific Cell Populations Deriving from Differentiated Human Embryonic Stem Cells

NARCIS (Netherlands)

Mengarelli, Isabella; Fryga, Andrew; Barberi, Tiziano

2016-01-01

Flow Cytometry-Sorting (FCM-Sorting) is a technique commonly used to identify and isolate specific types of cells from a heterogeneous population of live cells. Here we describe a multicolor flow cytometry technique that uses five distinct cell surface antigens to isolate four live populations with

Radon-induced DNA damage and apoptosis analyzed by flow cytometry

International Nuclear Information System (INIS)

Meenakshi, C.; Mohankumar, Mary N.

2012-01-01

Natural radiation is the major source of human exposure to ionizing radiation and its largest contributing component to effective doses arises from inhalation of 222 Rn and its radioactive progeny. 222 Rn, a chemically inert gas produced naturally from radium in rocks and soil is a proven source of lung cancer especially in closed environments such as mines and in poorly ventilated homes. Much of the data on the effect of radon in humans comes from epidemiological studies, often masked by confounding factors such as age, smoking and lifestyle. Radiation carcinogenesis is initiated by DNA damage and flow cytometry is a versatile, fast and accurate technique for the analysis of DNA damage as it offers the analysis of high number of individual cells in few minutes. An attempt was made to detect DNA damage and apoptosis after exposing human blood cells in vitro to radon by flow cytometry. Blood samples were collected from apparently healthy individuals and exposed in vitro to radon ranging between 1-5 mGy using a simple, portable irradiation assembly designed and tested at the Radiological Safety Division of Indira Gandhi Centre for Atomic Research. Cultures were initiated by the addition of phytohemagglutinin and cells were processed stained and analyzed for DNA damage and apoptosis by flow cytometry. CV values indicative of DNA damage were plotted against dose and were observed to increase in a dose dependent manner 3h after of irradiation. However no such response was observed at 24h and 48h. Nevertheless, the percentage of apoptotic cells increased steadily with dose after 24 and 48h post exposure. DNA breaks appear to be rejoined after about 24h of irradiation. However apoptotic cells increased with time and dose, suggesting elimination of highly damaged cells. Further experiments are needed to identify apoptotic cells as a biomarker of radiation exposure and risk. (author)

The use of flow cytometry to monitor chitin synthesis in regenerating protoplasts of Candida albicans.

Science.gov (United States)

Hector, R F; Braun, P C; Hart, J T; Kamarck, M E

1990-01-01

Flow cytometry was used to monitor chitin synthesis in regenerating protoplasts of the yeast Candida albicans. Comparisons of cells stained with Calcofluor White, a fluorochrome with known affinity for chitin, and cells incubated in the presence of N-[3H]-acetylglucosamine, the precursor substrate for chitin, showed a linear relationship between fluorescence and incorporation of label over time. Changes in both the fluorescence and light scatter of regenerating protoplasts treated with inhibitors of fungal chitin synthase were also quantitated by flow cytometry.

Radiation-induced apoptosis in thymocytes as determined by flow cytometry

International Nuclear Information System (INIS)

Su Xu; Zhang Yingchun; Liu Shuzheng

1995-01-01

Programmed cell death (PCD), or apoptosis, is a conceptually different way of cell death from necrosis. PCD plays an important role in immunologic regulation, PCD in thymocytes was analyzed by flow cytometry following in vitro X-irradiation. It was found that culturing of thymocytes could induce PCD which showed a time dependent increase. Four hours after culturing, 16% of thymocytes was found in the Ao region (PCD is shown in the Ao region of the histogram of flow cytometry). PCD in thymocytes showed a time dependent increase after 2.0 Gy X-irradiation, being significantly higher than that in the control at the same culturing time. 24 hours after X-irradiation in vitro, it was found that with doses below 100 mGy PCD was not significantly different from the control at the same culturing time. But when the doses were above 100 mGy, PCD showed a dose dependent increase, being significantly higher than that of the control at the same culturing time. These results are important in the understanding of the biological effects of low dose radiation

Immune Response to Mycobacterial Infection: Lessons from Flow Cytometry

OpenAIRE

Rovina, Nikoletta; Panagiotou, Marios; Pontikis, Konstantinos; Kyriakopoulou, Magdalini; Koulouris, Nikolaos G.; Koutsoukou, Antonia

2013-01-01

Detecting and treating active and latent tuberculosis are pivotal elements for effective infection control; yet, due to their significant inherent limitations, the diagnostic means for these two stages of tuberculosis (TB) to date remain suboptimal. This paper reviews the current diagnostic tools for mycobacterial infection and focuses on the application of flow cytometry as a promising method for rapid and reliable diagnosis of mycobacterial infection as well as discrimination between active...

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

Science.gov (United States)

Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

2016-01-01

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

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Yong Xue

Full Text Available Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

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....

A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

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Bendinelli Mauro

2009-03-01

Full Text Available Abstract Background Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with 51chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry. Results The method consists of culturing skin fibroblasts, immortalizing them with a replication defective clone of simian virus 40, and finally transducing them with a bicistronic vector encoding the target antigen and the reporter green fluorescent protein. When used in a flow cytometry-based assay, the target cells obtained with this method proved valuable for assessing the viral envelope protein specific cytotoxic T lymphocyte activity in domestic cats acutely or chronically infected with feline immunodeficiency virus, a lentivirus similar to human immunodeficiency virus and used as animal model for AIDS studies. Conclusion Given the versatility of the bicistronic vector used, its ability to deliver multiple and large transgenes in target cells, and its extremely wide cell specificity when pseudotyped with the vesicular stomatitis virus envelope protein, the method is potentially exploitable in many animal species.

A Flow Cytometry Protocol to Estimate DNA Content in the Yellowtail Tetra Astyanax altiparanae

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Pedro L. P. Xavier

2017-09-01

Full Text Available The production of triploid yellowtail tetra Astyanax altiparanae is a key factor to obtain permanently sterile individuals by chromosome set manipulation. Flow cytometric analysis is the main tool for confirmation of the resultant triploids individuals, but very few protocols are specific for A. altiparanae species. The current study has developed a protocol to estimate DNA content in this species. Furthermore, a protocol for long-term storage of dorsal fins used for flow cytometry analysis was established. The combination of five solutions with three detergents (Nonidet P-40 Substitute, Tween 20, and Triton X-100 at 0.1, 0.2, and 0.4% concentration was evaluated. Using the best solution from this first experiment, the addition of trypsin (0.125, 0.25, and 0.5% and sucrose (74 mM and the effects of increased concentrations of the detergents at 0.6 and 1.2% concentration were also evaluated. After adjustment of the protocol for flow cytometry, preservation of somatic tissue or isolated nuclei was also evaluated by freezing (at −20°C and fixation in saturated NaCl solution, acetic methanol (1:3, ethanol, and formalin at 10% for 30 or 60 days of storage at 25°C. Flow cytometry analysis in yellowtail tetra species was optimized using the following conditions: lysis solution: 9.53 mM MgCl2.7H20; 47.67 mM KCl; 15 mM Tris; 74 mM sucrose, 0.6% Triton X-100, pH 8.0; staining solution: Dulbecco's PBS with DAPI 1 μg mL−1; preservation procedure: somatic cells (dorsal fin samples frozen at −20°C. Using this protocol, samples may be stored up to 60 days with good accuracy for flow cytometry analysis.

Preparation of rat islet B-cell-enriched fractions by light-scatter flow cytometry

International Nuclear Information System (INIS)

Rabinovitch, A.; Russell, T.; Shienvold, F.; Noel, J.; Files, N.; Patel, Y.; Ingram, M.

1982-01-01

Flow cytometry has been examined as a method to separate islet cells into homogeneous subpopulations. Collagenase-isolated rat islets were dissociated into single cells and these were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. Light scatter histograms showed two peaks of viable cells. Radioimmunoassay of hormone content in cell fractions collected across the the two peaks showed that glucagon-containing cells were concentrated towards the left side of the left peak and somatostatin-containing cells were concentrated towards the right side of the left peak, whereas insulin-containing cells were clearly enriched in the right peak. The B-cell-enriched fraction (90% B cells, 3% A cells, 2% D cells) exhibited significant insulin secretory responses to glucose (16.7 mM), and 3-isobutyl-1-methylxanthine (0.1 mM), during a 24-h culture period, and these responses were slightly greater than those observed in the original mixed islet cell preparation (66% B cells, 14% A cells, and 4% D cells). These results indicate that flow cytometry can be applied to sort pancreatic islet cells into populations enriched in specific endocrine cell types for further study of the functions of individual cell types

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.

Cytotoxicity and genotoxicity of intravitreal adalimumab administration in rabbit retinal cells

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Álcio Coutinho de Paula

2015-04-01

Full Text Available Purpose: To assess the cytotoxicity and genotoxicity of intravitreal adalimumab treatment in an animal experimental model using cytological and molecular techniques. Methods: Eighteen rabbits were randomly assigned to three groups: control, adalimumab treatment, and placebo. Cytotoxicity on retinal cells was evaluated using flow cytometry assays to determine the level of apoptosis and necrosis. Genotoxicity was evaluated by comet assays to assess DNA damage, and quantitative real-time polymerase chain reaction (qPCR was used to evaluate expression of apoptosis-inducing caspases (8 and 3. Results: No cytotoxicity or genotoxicity was observed in any of the two treatment groups (adalimumab and placebo following intravitreal administration compared with the control group. Flow cytometry analysis revealed that more than 90% of the cells were viable, and only a low proportion of retinal cells presented apoptotic (~10% or necrotic (<1% activity across all groups. Molecular damage was also low with a maximum of 6.4% DNA degradation observed in the comet assays. In addition, no increase in gene expression of apoptosis-inducing caspases was observed on retinal cells by qPCR in both the adalimumab and placebo groups compared with the control group. Conclusion: The use of adalimumab resulted in no detectable cytotoxicity or genotoxicity on retinal cells for up to 60 days upon administration. These results therefore indicate that adalimumab may be a safe option for intravitreal application to treat ocular inflammatory diseases in which TNF-α is involved.

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry

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Crucian, B. E.; Stowe, R. P.; Pierson, D. L.; Sams, C. F.

2001-01-01

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may

Polyploidy in the Olive Complex (Olea europaea): Evidence from Flow Cytometry and Nuclear Microsatellite Analyses

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Besnard, G.; Garcia-Verdugo, C.; Rubio De Casas, R.; Treier, U. A.; Galland, N.; Vargas, P.

2008-01-01

Background Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. Methods Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. Key Results Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. Conclusions This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors

Monitoring hyperproliferative disorders in human skin: flow cytometry of changing cytokeratin expression.

NARCIS (Netherlands)

Franssen, M.E.J.; Boezeman, J.B.M.; Kerkhof, P.C.M. van de; Erp, P.E.J. van

2004-01-01

BACKGROUND: Monitoring dynamics of different cell populations in solid tissues using flow cytometry has several limitations. The interaction and changes in epidermal subpopulations in hyperproliferative skin disorders such as psoriasis, a very common chronic inflammatory skin disease, may, however,

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

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Zhou, Gang [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China); Liu, Naicheng; Wang, Zhenheng [Nanjing University, Department of Orthopedics, Jinling Hospital, School of Medicine (China); Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng, E-mail: jfzhang@nju.edu.cn [Nanjing University, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences (China)

2017-02-15

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

Quantitative analysis of gold and carbon nanoparticles in mammalian cells by flow cytometry light scattering

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Zhou, Gang; Liu, Naicheng; Wang, Zhenheng; Shi, Tongguo; Gan, Jingjing; Wang, Zhenzhen; Zhang, Junfeng

2017-02-01

Nanoparticle-based applications for diagnostics and therapeutics have been extensively studied. These applications require a profound understanding of the fate of nanoparticles (NPs) in cellular environments. However, until now, few analytical methods are available and most of them rely on fluorescent properties or special elements of NPs; therefore, for NPs without observable optical properties or special elements, the existing methods are hardly applicable. In this study, we introduce a flow cytometry light scattering (FCLS)-based approach that quantifies in situ NPs accurately in mammalian cells. Continuous cells of heterogeneous human epithelial colorectal adenocarcinoma (Caco-2 cells), mouse peritoneal macrophages (MPM), and human adenocarcinomic alveolar basal epithelia (A549 cells) were cultured with NPs with certain concentrations and size. The intensity of the flow cytometric side scattered light, which indicates the quantity of NPs in the cells, was analyzed. The result shows an accurate size- and dose-dependent uptake of Au NPs (5, 30, 250 nm) in Caco-2 cells. The size- and dose- dependence of Au NPs (5, 30, 250 nm) and carbon NPs (50, 500 nm) in cells was validated by transmission electron microscope (TEM). This paper demonstrates the great potential of flow cytometry light scattering in the quantitative study of the size and dose effect on in situ metallic or non-metallic NPs in mammalian cells.

Hyperchromatic laser scanning cytometry

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Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

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

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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...

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

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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.

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

Analysis of Cellular DNA Content by Flow Cytometry.

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Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

[Clinical usefulness of urine-formed elements' information obtained from bacteria detection by flow cytometry method that uses nucleic acid staining].

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Nakagawa, Hiroko; Yuno, Tomoji; Itho, Kiichi

2009-03-01

Recently, specific detection method for Bacteria, by flow cytometry method using nucleic acid staining, was developed as a function of automated urine formed elements analyzer for routine urine testing. Here, we performed a basic study on this bacteria analysis method. In addition, we also have a comparison among urine sediment analysis, urine Gram staining and urine quantitative cultivation, the conventional methods performed up to now. As a result, the bacteria analysis with flow cytometry method that uses nucleic acid staining was excellent in reproducibility, and higher sensitivity compared with microscopic urinary sediment analysis. Based on the ROC curve analysis, which settled urine culture method as standard, cut-off level of 120/microL was defined and its sensitivity = 85.7%, specificity = 88.2%. In the analysis of scattergram, accompanied with urine culture method, among 90% of rod positive samples, 80% of dots were appeared in the area of 30 degrees from axis X. In addition, one case even indicated that analysis of bacteria by flow cytometry and scattergram of time series analysis might be helpful to trace the progress of causative bacteria therefore the information supposed to be clinically significant. Reporting bacteria information with nucleic acid staining flow cytometry method is expected to contribute to a rapid diagnostics and treatment of urinary tract infections. Besides, the contribution to screening examination of microbiology and clinical chemistry, will deliver a more efficient solution to urine analysis.

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry.

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Tang, Vera A; Renner, Tyler M; Fritzsche, Anna K; Burger, Dylan; Langlois, Marc-André

2017-12-19

Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cell-derived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.

Internalisation of polymeric nanosensors in mesenchymal stem cells: analysis by flow cytometry and confocal microscopy.

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Coupland, Paul G; Fisher, Karen A; Jones, D Rhodri E; Aylott, Jonathan W

2008-09-10

The aim of this study was to demonstrate that flow cytometry and confocal microscopy could be applied in a complementary manner to analyse the internalisation of polymeric nanosensors in mesenchymal stem cells (MSC). The two techniques are able to provide en masse data analysis of nanosensors from large cell populations and detailed images of intracellular nanosensor localisation, respectively. The polyacrylamide nanosensors used in this investigation had been modified to contain free amine groups which were subsequently conjugated to Tat peptide, which acted as a delivery vector for nanosensor internalisation. Flow cytometry was used to confirm the health of MSC culture and assess the impact of nanosensor internalisation. MSC were characterised using fluorescently tagged CD cell surface markers that were also used to show that nanosensor internalisation did not negatively impact on MSC culture. Additionally it was shown that flow cytometry can be used to measure fluorophores located both on the cell surface and internalised within the cell. Complementary data was obtained using confocal microscopy to confirm nanosensor internalisation within MSC.

CytometryML and other data formats

Science.gov (United States)

Leif, Robert C.

2006-02-01

Cytology automation and research will be enhanced by the creation of a common data format. This data format would provide the pathology and research communities with a uniform way for annotating and exchanging images, flow cytometry, and associated data. This specification and/or standard will include descriptions of the acquisition device, staining, the binary representations of the image and list-mode data, the measurements derived from the image and/or the list-mode data, and descriptors for clinical/pathology and research. An international, vendor-supported, non-proprietary specification will allow pathologists, researchers, and companies to develop and use image capture/analysis software, as well as list-mode analysis software, without worrying about incompatibilities between proprietary vendor formats. Presently, efforts to create specifications and/or descriptions of these formats include the Laboratory Digital Imaging Project (LDIP) Data Exchange Specification; extensions to the Digital Imaging and Communications in Medicine (DICOM); Open Microscopy Environment (OME); Flowcyt, an extension to the present Flow Cytometry Standard (FCS); and CytometryML. The feasibility of creating a common data specification for digital microscopy and flow cytometry in a manner consistent with its use for medical devices and interoperability with both hospital information and picture archiving systems has been demonstrated by the creation of the CytometryML schemas. The feasibility of creating a software system for digital microscopy has been demonstrated by the OME. CytometryML consists of schemas that describe instruments and their measurements. These instruments include digital microscopes and flow cytometers. Optical components including the instruments' excitation and emission parts are described. The description of the measurements made by these instruments includes the tagged molecule, data acquisition subsystem, and the format of the list-mode and/or image data. Many

Flow Cytometry Method as a Diagnostic Tool for Pleural Fluid Involvement in a Patient with Multiple Myeloma

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MUZAFFER KEKLIK

2012-01-01

Full Text Available

Multiple myeloma is a malignant proliferation of plasma cells that mainly affects bone marrow. Pleural effusions secondary to pleural myelomatous involvement have rarely been reported in the literature. As it is rarely detected, we aimed to report a case in which pleural effusion of a multiple myeloma was confirmed as true myelomatous involvement by flow cytometry method. A 52-years old man presented to our clinic with chest and back pain lasting for 3 months. On the chest radiography, pleural fluid was detected in left hemithorax. Pleural fluid flow cytometry was performed. In the flow cytometry, CD56, CD38 and CD138 found to be positive, while CD19 was negative. True myelomatous pleural effusions are very uncommon, with fewer than 100 cases reported worldwide. Flow cytometry is a potentially useful diagnostic tool for clinical practice. We presented our case; as it has been rarely reported, although flow cytometer is a simple method for detection of pleural fluid involvement in multiple myeloma.

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

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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.

Curcumin-Combretastatin Nanocells as Breast Cancer Cytotoxic and Antiangiogenic Agent

Science.gov (United States)

2008-09-01

encapsulation of curcumin to increase its solubility and cytotoxicity. 5 ImageStream multispectral imaging flow cytometry and a real-time cell...easily soluble in water and most of consumed curcumin does not reach the bloodstream. In several Phase I clinical trials where patients were...that can render this hydrophobic polyphenol soluble in an aqueous phase medium. Recently, two liposomal formulations of curcumin have been reported

Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry

International Nuclear Information System (INIS)

Ross, Carley D.; French, C. Tenley; Keysar, Stephen B.; Fox, Michael H.

2007-01-01

We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A L ) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A L cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis

Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

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Berkay Saraymen

2016-03-01

Full Text Available Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immuno­logical methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in pa­tients diagnosed with hematologic malignancy. Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leuke­mia, and twenty healthy controls who were admitted to Erci­yes University Hematology-Oncology Hospital. The suspend­ed cells from bone marrow samples of patients and the pe­ripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry. Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lympho­blastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblas­tic leukemia and acute lymphoblastic leukemia bone marrow samples was reported. Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for mea­suring P-glycoprotein expression and suggests the pos­sibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

NARCIS (Netherlands)

Heijnen, I.; Barnett, D.; Arroz, M.J.; Barry, S.M.; Bonneville, M.; Brando, B.; D'Hautcourt, J.L.; Kern, F.; Totterman, T.H.; Marijt, E.W.; Bossy, D.; Preijers, F.W.M.B.; Rothe, G.; Gratama, J.W.

2004-01-01

BACKGROUND: HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic

The role of flow cytometry in the study of cell growth in the rat anterior pituitary gland

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M Vitale

2009-12-01

Full Text Available Flow cytometry is a suitable technique for studying in vivo and in vitro the cell cycle kinetics of different animal and human tissues, both in normal and tumoral conditions. The rat anterior pituitary gland is a model to investigate cell growth and replication of differentiated, neuroendocrine cells, and we report current evidence on its cell cycle kinetics as well as on the role played by flow cytometry in this type of study. The proliferation potential of normal anterior pituitary cells is related to a number of different conditions, including heterogeneity of cell types, age and sex of donors, and circadian influences. In addition, the trend of cell proliferation in both in vivo and in vitro studies is similar, suggesting that cultured anterior pituitary elements may, at least in parts, retain growth features analogous to those of the intact gland. Sorting of selective cell types and analysis of the relation between proliferating anterior pituitary cells and the light-dark cycle have shown that flow cytometry may be useful to investigate the replication process of the gland. By using a combination of flow cytometry, light microscopic immunocytochemistry and morphometry, we have reported a peculiar trend of proliferation in prima- ry monolayer cultures of rat anterior pituitary gland, characterized by a non-linear reduction in their proliferation rate with advancing age, primarily dependent on a reduced transition of cells from the G0/G1- to the early S-phase pool. These studies indicate that flow cytometry offers insights into cell cycle check points of anterior pituitary cells, and suggest that it might be applied to the study of growth of selective pituitary elements, both in normal and tumoral conditions.

Use of flow cytometry for the possible identification of radio-induced changes in DNA of animal cells

International Nuclear Information System (INIS)

Spano, M.; Leonardi, M.; Cordelli, E.

1991-01-01

Since DNA is the main cellular target of ionizing irradiation, methods fit for analyzing DNA alterations should be able to discern irradiated versus control cells. Flow cytometry allows the rapid measurement of DNA content of single chromosomes or cell nuclei at very high resolution on a statistically significant sample. Alterations of chromatine structure can also be analyzed by flow cytometry. Briefly, evaluation of in situ DNA resistance to denaturation can be evaluated by flow cytometric analysis of different staining pattern of single versus double strange regions of DNA. In the present work both approaches were used with the aim to recognize cells derived from an irradiated sample of breast chicken. Although flow cytometry has been demonstrated to be a useful tool to detect DNA alterations and has been widely used to detect damages on DNA induced by several physical and chemical agents, it was unable to detect clastogenic effects induced by electrons on DNA of chicken breast cells. Heavily irradiated nuclei, even if challenged by denaturating treatments that partially collapse chromatine organization, do not present differences from non irradiated samples after flow cytometric DNA content measurement. (16 refs)

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

Energy Technology Data Exchange (ETDEWEB)

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).

Molecular Characterization of Gastric Epithelial Cells Using Flow Cytometry

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Kevin A. Bockerstett

2018-04-01

Full Text Available The ability to analyze individual epithelial cells in the gastric mucosa would provide important insight into gastric disease, including chronic gastritis and progression to gastric cancer. However, the successful isolation of viable gastric epithelial cells (parietal cells, neck cells, chief cells, and foveolar cells from gastric glands has been limited due to difficulties in tissue processing. Furthermore, analysis and interpretation of gastric epithelial cell flow cytometry data has been difficult due to the varying sizes and light scatter properties of the different epithelial cells, high levels of autofluorescence, and poor cell viability. These studies were designed to develop a reliable method for isolating viable single cells from the corpus of stomachs and to optimize analyses examining epithelial cells from healthy and diseased stomach tissue by flow cytometry. We performed a two stage enzymatic digestion in which collagenase released individual gastric glands from the stromal tissue of the corpus, followed by a Dispase II digestion that dispersed these glands into greater than 1 × 106 viable single cells per gastric corpus. Single cell suspensions were comprised of all major cell lineages found in the normal gastric glands. A method describing light scatter, size exclusion, doublet discrimination, viability staining, and fluorescently-conjugated antibodies and lectins was used to analyze individual epithelial cells and immune cells. This technique was capable of identifying parietal cells and revealed that gastric epithelial cells in the chronically inflamed mucosa significantly upregulated major histocompatibility complexes (MHC I and II but not CD80 or CD86, which are costimulatory molecules involved in T cell activation. These studies describe a method for isolating viable single cells and a detailed description of flow cytometric analysis of cells from healthy and diseased stomachs. These studies begin to identify effects of

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry

Science.gov (United States)

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856. PMID:29474436

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

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Muhammed Majeed

Full Text Available Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore® is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

Science.gov (United States)

Majeed, Muhammed; Majeed, Shaheen; Nagabhushanam, Kalyanam; Punnapuzha, Ardra; Philip, Sheena; Mundkur, Lakshmi

2018-01-01

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Reticulocyte analysis using flow cytometry.

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Corberand, J X

1996-12-01

Automation of the reticulocyte count by means of flow cytometry has considerably improved the quality of this investigation. This article deals firstly with the reasons for the poor performance of the microscopic technique and with the physiological principles underlying identification and classification of reticulocytes using RNA labeling. It then outlines the automated methods currently on the market, which can be classified in three categories: a) "general-purpose" cytofluorometers, which in clinical laboratories usually deal with lymphocyte immunophenotyping; b) the only commercially available cytofluorometer dedicated to the reticulocyte count; this automat has the advantage of requiring no human intervention as it merely needs to be fed with samples; c) hematology analyzers with specific modules for automatic counting of reticulocytes previously incubated with a non-fluorescent dye. Of the various fluorescent markers available, thiazole orange, DEQTC iodide and auramine are most often used for this basic hematology test. The quality of the count, the availability of new reticulocyte indices (maturation index, percentage of young reticulocytes) and rapidity of the count give this test renewed value in the practical approach to the diagnosis of anemia, and also open new perspectives in the surveillance of aplastic anemia after chemotherapy or bone marrow grafting.

Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

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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.

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

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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.

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

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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.

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

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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...... 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...

Detection of microparticles from human red blood cells by multiparametric flow cytometry

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Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo

2015-01-01

Background During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as “storage lesions”. These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies. PMID:25369588

FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

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Poulton, Nicole J

2016-01-01

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Cytotoxic and biological effects of bulk fill composites on rat cortical neuron cells.

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Kamalak, Hakan; Kamalak, Aliye; Taghizadehghalehjoughi, Ali; Hacımüftüoğlu, Ahmet; Nalcı, Kemal Alp

2018-03-28

The aim of this study was to investigate potential cellular responses and biological effects of new generation dental composites on cortical neuron cells in two different exposure times. The study group included five different bulk-fill flow able composites; Surefil SDR Flow, X-tra Base Flow, Venus Bulk Flow, Filtek Bulk Flow and Tetric-Evo Flow. They were filled in Teflon molds (Height: 4 mm, Width: 6 mm) and irradiated for 20 s. Cortical neuron cells were inoculated into 24-well plates. After 80% of the wells were coated, the 3 µm membrane was inserted and dental filling materials were added. The experiment was continued for 24 and 72 h. Cell viability measured by MTT assay test, total antioxidant and total oxidant status were examined using real assay diagnostic kits. The patterns of cell death (apoptosis) were analyzed using annexin V-FITC staining with flow cytometry. Β-defensins were quantitatively assessed by RT-PCR. IL-6, IL-8 and IL-10 cytokines were measured from the supernatants. All composites significantly affected analyses parameters during the exposure durations. Our data provide evidence that all dental materials tested are cytotoxic in acute phase and these effects are induced cellular death after different exposure periods. Significant cytotoxicity was detected in TE, XB, SS, FBF and VBF groups at 24 and 72 h, respectively.

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

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Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

1999-01-01

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

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

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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

European canine lymphoma network consensus recommendations for reporting flow cytometry in canine hematopoietic neoplasms.

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Comazzi, S; Avery, P R; Garden, O A; Riondato, F; Rütgen, B; Vernau, W

2017-09-01

Flow cytometry (FC) is assuming increasing importance in diagnosis in veterinary oncology. The European Canine Lymphoma Network (ECLN) is an international cooperation of different institutions working on canine lymphoma diagnosis and therapy. The ECLN panel of experts on FC has defined the issue of reporting FC on canine lymphoma and leukemia as their first hot topic, since a standardized report that includes all the important information is still lacking in veterinary medicine. The flow cytometry panel of the ECLN started a consensus initiative using the Delphi approach. Clinicians were considered the main target of FC reports. A panel of experts in FC was interrogated about the important information needed from a report. Using the feedback from clinicians and subsequent discussion, a list of information to be included in the report was made, with four different levels of recommendation. The final report should include both a quantitative part and a qualitative or descriptive part with interpretation of the salient results. Other items discussed included the necessity of reporting data regarding the quality of samples, use of absolute numbers of positive cells, cutoff values, the intensity of fluorescence, and possible aberrant patterns of antigen expression useful from a clinical point of view. The consensus initiative is a first step toward standardization of diagnostic approach to canine hematopoietic neoplasms among different institutions and countries. This harmonization will improve communication and patient care and also facilitate the multicenter studies necessary to further our knowledge of canine hematopoietic neoplasms. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

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

Science.gov (United States)

2013-01-24

... need for such products to assist clinical laboratories in performing this testing. FDA has been working... this workshop include: (1) Overview of Quality control and standardization issues associated with Clinical Flow Cytometry (FCM), including discussion of a NIST traceable standard; (2) Biological controls...

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

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Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

EMS mutant spectra generated by multi-parameter flow cytometry

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Keysar, Stephen B. [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Fox, Michael H., E-mail: michael.fox@colostate.edu [Cell and Molecular Biology Graduate Program, Colorado State University, Fort Collins, CO (United States); Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO (United States)

2009-12-01

The CHO A{sub L} cell line contains a single copy of human chromosome 11 that encodes several cell surface proteins including glycosyl phosphatidylinositol (GPI) linked CD59 and CD90, as well as CD98, CD44 and CD151 which are not GPI-linked. The flow cytometry mutation assay (FCMA) measures mutations of the CD59 gene by the absence of fluorescence when stained with antibodies against the CD59 cell surface protein. We have measured simultaneous mutations in CD59, CD44, CD90, CD98 and CD151 to generate a mutant spectrum for ionizing radiation. After treatment with ethyl methanesulfonate (EMS) many cells have an intermediate level of CD59 staining. Single cells were sorted from CD59{sup -} regions with varying levels of fluorescence and the resulting clonal populations had a stable phenotype for CD59 expression. Mutant spectra were generated by flow cytometry using the isolated clones and nearly all clones were mutated in CD59 only. Interestingly, about 60% of the CD59 negative clones were actually GPI mutants determined by staining with the GPI specific fluorescently labeled bacterial toxin aerolysin (FLAER). The GPI negative cells are most likely caused by mutations in the X-linked pigA gene important in GPI biosynthesis. Small mutations of pigA and CD59 were expected for the alkylating agent EMS and the resulting spectra are significantly different than the large deletions found when analyzing radiation mutants. After analyzing the CD59{sup -} clonal populations we have adjusted the FCMA mutant regions from 1% to 10% of the mean of the CD59 positive peak to include the majority of CD59 mutants.

Flow cytometry-assisted rapid isolation of recombinant Plasmodium berghei parasites exemplified by functional analysis of aquaglyceroporin

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Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W.A.

2012-01-01

The most critical bottleneck in the generation of recombinant Plasmodium berghei parasites is the mandatory in vivo cloning step following successful genetic manipulation. This study describes a new technique for rapid selection of recombinant P. berghei parasites. The method is based on flow cytometry to isolate isogenic parasite lines and represents a major advance for the field, in that it will speed the generation of recombinant parasites as well as cut down on animal use significantly. High expression of GFP during blood infection, a prerequisite for robust separation of transgenic lines by flow cytometry, was achieved. Isogenic recombinant parasite populations were isolated even in the presence of a 100-fold excess of wild-type (WT) parasites. Aquaglyceroporin (AQP) loss-of-function mutants and parasites expressing a tagged AQP were generated to validate this approach. aqp− parasites grow normally within the WT phenotypic range during blood infection of NMRI mice. Similarly, colonization of the insect vector and establishment of an infection after mosquito transmission were unaffected, indicating that AQP is dispensable for life cycle progression in vivo under physiological conditions, refuting its use as a suitable drug target. Tagged AQP localized to perinuclear structures and not the parasite plasma membrane. We suggest that flow-cytometric isolation of isogenic parasites overcomes the major roadblock towards a genome-scale repository of mutant and transgenic malaria parasite lines. PMID:23137753

CytometryML with DICOM and FCS

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Leif, Robert C.

2018-02-01

Abstract: Flow Cytometry Standard, FCS, and Digital Imaging and Communications in Medicine standard, DICOM, are based on extensive, superb domain knowledge, However, they are isolated systems, do not take advantage of data structures, require special programs to read and write the data, lack the capability to interoperate or work with other standards and FCS lacks many of the datatypes necessary for clinical laboratory data. The large overlap between imaging and flow cytometry provides strong evidence that both modalities should be covered by the same standard. Method: The XML Schema Definition Language, XSD 1.1 was used to translate FCS and/or DICOM objects. A MIFlowCyt file was tested with published values. Results: Previously, a significant part of an XML standard based upon a combination of FCS and DICOM has been implemented and validated with MIFlowCyt data. Strongly typed translations of FCS keywords have been constructed in XML. These keywords contain links to their DICOM and FCS equivalents.

Comparative studies of cytotoxic and apoptotic properties of different extracts and the essential oil of Lavandula angustifolia on malignant and normal cells.

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Tayarani-Najaran, Zahra; Amiri, Atefeh; Karimi, Gholamreza; Emami, Seyed Ahmad; Asili, Javad; Mousavi, Seyed Hadi

2014-01-01

Lavender (Lavandula angustifolia Mill.) is a bush-like shrub from Lamiaceae. The herb has been used in alternative medicine for several centuries. In this study, the cytotoxicity and the mechanisms of cell death induced by 3 different extracts of aerial parts and the essential oil of L. angustifolia were compared in normal and cancerous human cells. Malignant (HeLa and MCF-7 cell lines) and nonmalignant (human fibroblasts) cells were incubated with different concentrations of the plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). The molecules as apoptotic signal translation, including Bax and cleaved PARP, were identified by Western blot. Ethanol and n-hexane extracts and essential oil exhibited significant cytotoxicity to malignant cells but marginal cytotoxicity to human fibroblasts in vitro and induced a sub-G1 peak in flow cytometry histogram of treated cells compared to the control. Western blot analysis demonstrated that EtOH and n-hexane extracts upregulated Bax expression, also it induced cleavage of PARP in HeLa cells compared to the control. In conclusion, L. angustifolia has cytotoxic and apoptotic effects in HeLa and MCF-7 cell lines, and apoptosis is proposed as the possible mechanism of action.

Preparation and flow cytometry of uniform silica-fluorescent dye microspheres.

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Bele, Marjan; Siiman, Olavi; Matijević, Egon

2002-10-15

Uniform fluorescent silica-dye microspheres have been prepared by coating preformed monodispersed silica particles with silica layers containing rhodamine 6G or acridine orange. The resulting dispersions exhibit intense fluorescent emission between 500 and 600 nm, over a broad excitation wavelength range of 460 to 550 nm, even with exceedingly small amounts of dyes incorporated into the silica particles (10-30 ppm, expressed as weight of dye relative to weight of dry particles). The fluorescent particles can be prepared in micrometer diameters suitable for analyses using flow cytometry with 488-nm laser excitation.

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

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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.

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...

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

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......  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......-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.  ...

Utility of peripheral blood immunophenotyping by flow cytometry in the diagnosis of pediatric acute leukemia.

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Metrock, Laura K; Summers, Ryan J; Park, Sunita; Gillespie, Scott; Castellino, Sharon; Lew, Glen; Keller, Frank G

2017-10-01

Childhood acute leukemia is traditionally diagnosed from a bone marrow aspirate (BMA). New-onset acute leukemia patients do not always have visible circulating blasts in the peripheral blood (PB) at diagnosis. While the role of bone marrow flow cytometry for the diagnosis of acute leukemia is well established, the utility of PB flow cytometry (PBFC) is unknown. We performed a single-institution retrospective analysis to compare PBFC versus BMA in establishing or excluding a diagnosis of childhood acute leukemia. We retrospectively identified 485 PBFC samples with concurrent BMA from 2008 to 2013. Results of four-color flow cytometry for immunophenotypic characterization of leukemic versus nonclonal disease were characterized. Sensitivity and specificity were calculated among patients without a known diagnosis or prior therapy. Among 485 samples eligible for analysis, 120 had negative PBFC and BMA, 359 had positive PBFC and BMA, 3 had negative PBFC and positive BMA, and 3 had positive PBFC and negative BMA. There were small but significant differences in sensitivity (100 vs. 93.8%; P = 0.002) and positive predictive value (100 vs. 93.8%; P = 0.002) favoring BMA over PBFC among those demonstrating absence of circulating morphologic blasts. PBFC has high sensitivity and specificity for the diagnosis of childhood acute leukemia. The predictive value of PBFC remains high for patients without visible circulating blasts and may enhance the diagnostic process for determining the indications for marrow testing. © 2017 Wiley Periodicals, Inc.

Highly multiparametric analysis by mass cytometry.

Science.gov (United States)

Ornatsky, Olga; Bandura, Dmitry; Baranov, Vladimir; Nitz, Mark; Winnik, Mitchell A; Tanner, Scott

2010-09-30

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays. Copyright © 2010 Elsevier B.V. All rights reserved.

A perspective for biomedical data integration: Design of databases for flow cytometry

Directory of Open Access Journals (Sweden)

Lakoumentas John

2008-02-01

Full Text Available Abstract Background The integration of biomedical information is essential for tackling medical problems. We describe a data model in the domain of flow cytometry (FC allowing for massive management, analysis and integration with other laboratory and clinical information. The paper is concerned with the proper translation of the Flow Cytometry Standard (FCS into a relational database schema, in a way that facilitates end users at either doing research on FC or studying specific cases of patients undergone FC analysis Results The proposed database schema provides integration of data originating from diverse acquisition settings, organized in a way that allows syntactically simple queries that provide results significantly faster than the conventional implementations of the FCS standard. The proposed schema can potentially achieve up to 8 orders of magnitude reduction in query complexity and up to 2 orders of magnitude reduction in response time for data originating from flow cytometers that record 256 colours. This is mainly achieved by managing to maintain an almost constant number of data-mining procedures regardless of the size and complexity of the stored information. Conclusion It is evident that using single-file data storage standards for the design of databases without any structural transformations significantly limits the flexibility of databases. Analysis of the requirements of a specific domain for integration and massive data processing can provide the necessary schema modifications that will unlock the additional functionality of a relational database.

Multiparametric flow cytometry in the diagnosis and characterization of low-grade pulmonary mucosa-associated lymphoid tissue lymphomas.

Science.gov (United States)

Zaer, F S; Braylan, R C; Zander, D S; Iturraspe, J A; Almasri, N M

1998-06-01

Primary mucosa associated lymphoid tissue (MALT) lymphomas are rare neoplasms that seem to have a better prognosis than nodal lymphomas. Morphologic diagnosis of these lesions may be difficult because of features that overlap with those of benign lymphoid infiltrates. In this study, we assessed the contribution of multi-parametric flow cytometry in demonstrating clonality and further characterizing pulmonary MALT lymphomas. Based on a clinical or pathologic suspicion of MALT-lymphoma, 3 transbronchial biopsies, 4 fine needle aspirates, 1 core needle biopsy, and 13 wedge excisions of lung were submitted fresh (unfixed) to our laboratory for evaluation. Among the 13 cases diagnosed as MALT lymphomas, B-cell monoclonality was established by identifying expression of a single immunoglobulin light chain on CD20 or CD19-positive cells in 12 cases. One case lacked expression of both light chains on B-cells. Of 11 lymphoma cases in which CD5 and CD10 surface antigens were assessed, no cases expressed CD10, and 1 case demonstrated weak CD5 expression. Nine of 10 cases studied were diploid and 1 case was hyperdiploid. All of the lymphomas displayed low (< or = 3%) S-phase fractions consistent with low grade processes. In 10 patients with short follow-up, none died of their disease and the majority had no evidence of lymphoma dissemination. In seven of the remaining eight cases, B-cells were polyclonal consistent with reactive processes. In one morphologically reactive case, flow cytometric analysis was unsuccessful because of poor cell viability. The pulmonary MALT lymphomas in this study represent a group of B-cell tumors with distinctive morphologic, immunophenotypic, and cell kinetic characteristics. Multi-parametric flow cytometry is useful for confirming B-cell monoclonality and illustrating an antigenic profile compatible with this diagnosis. Flow cytometry can be particularly helpful when working with small biopsies and cytologic samples with limited diagnostic

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

Science.gov (United States)

Sipol, Alexandra A; Babenko, Elena V; Borisov, Vyacheslav I; Naumova, Elena V; Boyakova, Elena V; Yakunin, Dimitry I; Glazanova, Tatyana V; Chubukina, Zhanna V; Pronkina, Natalya V; Popov, Alexander M; Saveliev, Leonid I; Lugovskaya, Svetlana A; Lisukov, Igor A; Kulagin, Alexander D; Illingworth, Andrea J

2015-01-01

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.

Detection of mycoplasmas in goat milk by flow cytometry.

Science.gov (United States)

Assunção, Patricia; Davey, Hazel M; Rosales, Ruben S; Antunes, Nuno T; de la Fe, Christian; Ramirez, Ana S; de Galarreta, Carlos M Ruiz; Poveda, Jose B

2007-12-01

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples. (c) 2007 International Society for Analytical Cytology

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

International Nuclear Information System (INIS)

Santos, Neyliane Goncalves dos; Amaral, Ademir; Cavalcanti, Mariana Brayner . E-mail; Neves, Maria Amelia Batista; Machado, Cintia Gonsalves de Faria

2008-01-01

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)

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)

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

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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.

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

DEFF Research Database (Denmark)

Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

2013-01-01

to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis...

Sensitivity of hemozoin detection by automated flow cytometry in non- and semi-immune malaria patients

NARCIS (Netherlands)

Grobusch, Martin P.; Hänscheid, Thomas; Krämer, Benedikt; Neukammer, Jörg; May, Jürgen; Seybold, Joachim; Kun, Jürgen F. J.; Suttorp, Norbert

2003-01-01

BACKGROUND: Cell-Dyn automated blood cell analyzers use laser flow cytometry technology, allowing detection of malaria pigment (hemozoin) in monocytes. We evaluated the value of such an instrument to diagnose malaria in febrile travelers returning to Berlin, Germany, the relation between the

CytometryML: a data standard which has been designed to interface with other standards

Science.gov (United States)

Leif, Robert C.

2007-02-01

Because of the differences in the requirements, needs, and past histories including existing standards of the creating organizations, a single encompassing cytology-pathology standard will not, in the near future, replace the multiple existing or under development standards. Except for DICOM and FCS, these standardization efforts are all based on XML. CytometryML is a collection of XML schemas, which are based on the Digital Imaging and Communications in Medicine (DICOM) and Flow Cytometry Standard (FCS) datatypes. The CytometryML schemas contain attributes that link them to the DICOM standard and FCS. Interoperability with DICOM has been facilitated by, wherever reasonable, limiting the difference between CytometryML and the previous standards to syntax. In order to permit the Resource Description Framework, RDF, to reference the CytometryML datatypes, id attributes have been added to many CytometryML elements. The Laboratory Digital Imaging Project (LDIP) Data Exchange Specification and the Flowcyt standards development effort employ RDF syntax. Documentation from DICOM has been reused in CytometryML. The unity of analytical cytology was demonstrated by deriving a microscope type and a flow cytometer type from a generic cytometry instrument type. The feasibility of incorporating the Flowcyt gating schemas into CytometryML has been demonstrated. CytometryML is being extended to include many of the new DICOM Working Group 26 datatypes, which describe patients, specimens, and analytes. In situations where multiple standards are being created, interoperability can be facilitated by employing datatypes based on a common set of semantics and building in links to standards that employ different syntax.

Evaluation of CD307a expression patterns during normal B-cell maturation and in B-cell malignancies by flow cytometry.

Science.gov (United States)

Auat, Mariangeles; Cardoso, Chandra Chiappin; Santos-Pirath, Iris Mattos; Rudolf-Oliveira, Renata Cristina Messores; Matiollo, Camila; Lange, Bárbara Gil; da Silva, Jessica Pires; Dametto, Gisele Cristina; Pirolli, Mayara Marin; Colombo, Maria Daniela Holthausen Perico; Santos-Silva, Maria Claudia

2018-02-24

Flow cytometric immunophenotyping is deemed a fundamental tool for the diagnosis of B-cell neoplasms. Currently, the investigation of novel immunophenotypic markers has gained importance, as they can assist in the precise subclassification of B-cell malignancies by flow cytometry. Therefore, the purpose of the present study was to evaluate the expression of CD307a during normal B-cell maturation and in B-cell malignancies as well as to investigate its potential role in the differential diagnosis of these entities. CD307a expression was assessed by flow cytometry in normal precursor and mature B cells and in 115 samples collected from patients diagnosed with precursor and mature B-cell neoplasms. CD307a expression was compared between neoplastic and normal B cells. B-acute lymphoblastic leukemia cases exhibited minimal expression of CD307a, displaying a similar expression pattern to that of normal B-cell precursors. Mantle cell lymphoma (MCL) cases showed the lowest levels of CD307a among mature B-cell neoplasms. CD307a expression was statistically lower in MCL cases than in chronic B lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL) cases. No statistical differences were observed between CD307a expression in neoplastic and normal plasma cells. These results indicate that the assessment of CD307a expression by flow cytometry could be helpful to distinguish CLL from MCL, and the latter from MZL. Although these results are not entirely conclusive, they provide a basis for further studies in a larger cohort of patients. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Flow cytometry of nucleated red blood cells used as monitoring technique for aquatic risk assessment. A review.

Directory of Open Access Journals (Sweden)

Bratosin D.

2016-05-01

Full Text Available During the last decades anthropogenic factors led to a significant enhancement of pollutants in aquatic environment and for several years, chemicals analysis has been commonly employed. These techniques cannot detect and quantify many environmental phenomena such as bioavailability, bioaccumulation and synergistic effects. For these reasons, many investigations for evaluating the effects of xenobiotic on organisms use in vitro or in vivo bioassays. The bioassays give a global response for all chemicals present in the environment and these represent one of the best ways to estimate the risk assessment of pollutants in environment for monitoring. For assessing cytotoxicity or ecotoxicity of pollutants (heavy metals, nanoparticles, etc. and to assess aquatic pollution degree and biomonitoring of Danube River and Danube Delta, we developed a new experimental cell system based on the apoptosis of nucleated erythrocytes from fishes and batrachians which are directly exposed to pollutants absorbed by different ways. Despite their structural simplicity, the erythrocytes of lower vertebrates preserve nucleus and mitochondria, both the sensors of the programmed cell death (PCD machinery to develop an apoptosis phenomenon. Our proposed bioassays which are based on the apoptosis phenomenon as induced biomarker by pollutants on fish or amphibians erythrocytes, evidenced by flow cytometry (apoptosis/necrosis discriminated by FITC-annexin-V labeling/PI and cellular viability measured with calcein-AM method could be rapid and very sensitive tests for in laboratory aquatic risk assessment and biomonitoring. Standardization and application of these tests will surely provide the opportunity of their use easily in ecotoxicological laboratories, biomonitoring of large river basins such as the Danube River Basin and will be also able deliver information on fish as a food product.

A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery.

Science.gov (United States)

Joslin, John; Gilligan, James; Anderson, Paul; Garcia, Catherine; Sharif, Orzala; Hampton, Janice; Cohen, Steven; King, Miranda; Zhou, Bin; Jiang, Shumei; Trussell, Christopher; Dunn, Robert; Fathman, John W; Snead, Jennifer L; Boitano, Anthony E; Nguyen, Tommy; Conner, Michael; Cooke, Mike; Harris, Jennifer; Ainscow, Ed; Zhou, Yingyao; Shaw, Chris; Sipes, Dan; Mainquist, James; Lesley, Scott

2018-05-01

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

Energy Technology Data Exchange (ETDEWEB)

Duan, Nuo; Wu, Shijia [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Yu, Ye [Zhangjiagang Entry-Exit Inspection and Quarantine Bureau, Zhangjiangang 215600 (China); Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China); Wang, Zhouping, E-mail: wangzp@jiangnan.edu.cn [State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122 (China)

2013-12-04

Graphical abstract: -- Highlights: •Two bacteria were simultaneously detected using QD-apt as labels by flow cytometry. •QD-apt were used for recognition and fluorescence detection of two bacteria. •The method was applied successfully for bacteria detection in real samples. -- Abstract: A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry.

A dual-color flow cytometry protocol for the simultaneous detection of Vibrio parahaemolyticus and Salmonella typhimurium using aptamer conjugated quantum dots as labels

International Nuclear Information System (INIS)

Duan, Nuo; Wu, Shijia; Yu, Ye; Ma, Xiaoyuan; Xia, Yu; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2013-01-01

Graphical abstract: -- Highlights: •Two bacteria were simultaneously detected using QD-apt as labels by flow cytometry. •QD-apt were used for recognition and fluorescence detection of two bacteria. •The method was applied successfully for bacteria detection in real samples. -- Abstract: A sensitive, specific method for the collection and detection of pathogenic bacteria was demonstrated using quantum dots (QDs) as a fluorescence marker coupled with aptamers as the molecular recognition element by flow cytometry. The aptamer sequences were selected using a bacterium-based SELEX strategy in our laboratory for Vibrio parahaemolyticus and Salmonella typhimurium that, when applied in this method, allows for the specific recognition of the bacteria from complex mixtures including shrimp samples. Aptamer-modified QDs (QD-apt) were employed to selectively capture and simultaneously detect the target bacteria with high sensitivity using the fluorescence of the labeled QDs. The signal intensity is amplified due to the high photostability of QDs nanoparticles, resulting in improved sensitivity over methods using individual dye-labeled probes. This proposed method is promising for the sensitive detection of other pathogenic bacteria in food stuff if suitable aptamers are chosen. The method may also provide another potential platform for the application of aptamer-conjugated QDs in flow cytometry

Cytotoxicity of the coagulant Moringa oleifera lectin (cMoL) to B16-F10 melanoma cells.

Science.gov (United States)

de Andrade Luz, Luciana; Rossato, Franco Aparecido; Costa, Rute Alves Pereira E; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; Coelho, Luana Cassandra Breitenbach Barroso

2017-10-01

Moringa oleifera seeds are used in alternative medicine to treat inflammation, tumors and bacterial and protozoan infections, for example. The seeds contain lectins, which are carbohydrate-binding proteins with several biological properties including cytotoxicity to cancer cells. In this work, we examined the cytotoxicity of the coagulant M. oleifera lectin (cMoL) on B16-F10 murine melanoma cells. cMoL cytotoxic effects were evaluated through trypan blue assay and flow cytometry analysis. Mitochondrial superoxide levels and activation of caspases 3, 8 and 9 were measured. cMoL (1.5-16μM) reduced viability and caused cell death of B16-F10 cells with an IC 50 of 9.72μM. Flow cytometry analysis indicated induction of necrosis and suggested the presence of cells in late apoptosis. Specificity for tumor cells was observed since death of normal human fibroblasts (GN) was not higher than 20% in treatments with cMoL from 1.5 to 16μM. Microscopy images revealed rounded shape and reduction of volume in B16-F10 cells treated with cMoL. cMoL increased mitochondrial ROS production and promoted caspases 3, 8 and 9 activation in B16-F10 cells, indicating the activation of apoptosis-related pathway. In conclusion, this study demonstrates that cMoL is cytotoxic to B16-F10 cells, which stimulates more investigation on the anticancer potential of this lectin. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Czech Academy of Sciences Publication Activity Database

Loureiro, J.; Pinto, G.; Lopes, T.; Doležel, Jaroslav; Santos, C.

2005-01-01

Roč. 221, - (2005), s. 815-822 ISSN 0032-0935 Institutional research plan: CEZ:AV0Z50380511 Keywords : Flow cytometry * ploidy stability * nuclear DNA content Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.108, year: 2005

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Science.gov (United States)

Chioccioli, Maurizio; Hankamer, Ben; Ross, Ian L.

2014-01-01

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth. PMID

Identification of a murine erythroblast subpopulation enriched in enucleating events by multi-spectral imaging flow cytometry.

Science.gov (United States)

Konstantinidis, Diamantis G; Pushkaran, Suvarnamala; Giger, Katie; Manganaris, Stefanos; Zheng, Yi; Kalfa, Theodosia A

2014-06-06

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters "aspect ratio" of a cell in the bright-field channel that aids in the recognition of elongated cells and "delta centroid XY Ter119/Draq5" that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

Combination of CD157 and FLAER to Detect Peripheral Blood Eosinophils by Multiparameter Flow Cytometry.

Science.gov (United States)

Carulli, Giovanni; Marini, Alessandra; Sammuri, Paola; Domenichini, Cristiana; Ottaviano, Virginia; Pacini, Simone; Petrini, Mario

2015-01-01

The identification of eosinophils by flow cytometry is difficult because most of the surface antigens expressed by eosinophils are shared with neutrophils. Some methods have been proposed, generally based on differential light scatter properties, enhanced autofluorescence, lack of CD16 or selective positivity of CD52. Such methods, however, show several limitations. In the present study we report a novel method based on the analysis of glycosylphosphatidylinositol (GPI)-linked molecules. The combination of CD157 and FLAER was used, since FLAER recognizes all GPI-linked molecules, while CD157 is absent on the membrane of eosinophils and expressed by neutrophils. Peripheral blood samples from normal subjects and patients with variable percentages of eosinophils (n = 31), and without any evidence for circulating immature myeloid cells, were stained with the combination of FLAER-Alexa Fluor and CD157-PE. A FascCanto II cytometer was used. Granulocytes were gated after CD33 staining and eosinophils were identified as CD157(-)/FLAER(+) events. Neutrophils were identified as CD157(+)/FLAER(+) events. The percentages of eosinophils detected by this method showed a very significant correlation both with automated counting and with manual counting (r = 0.981 and 0.989, respectively). Sorting assays were carried out by a S3 Cell Sorter: cytospins obtained from CD157(-)/FLAER(+) events consisted of 100% eosinophils, while samples from CD157(+)/FLAER(+) events were represented only by neutrophils. In conclusion, this method shows high sensitivity and specificity in order to distinguish eosinophils from neutrophils by flow cytometry. However, since CD157 is gradually up-regulated throughout bone marrow myeloid maturation, our method cannot be applied to cases characterized by immature myeloid cells.

Carboxylated nanodiamonds are neither cytotoxic nor genotoxic on liver, kidney, intestine and lung human cell lines.

Science.gov (United States)

Paget, V; Sergent, J A; Grall, R; Altmeyer-Morel, S; Girard, H A; Petit, T; Gesset, C; Mermoux, M; Bergonzo, P; Arnault, J C; Chevillard, S

2014-08-01

Although nanodiamonds (NDs) appear as one of the most promising nanocarbon materials available so far for biomedical applications, their risk for human health remains unknown. Our work was aimed at defining the cytotoxicity and genotoxicity of two sets of commercial carboxylated NDs with diameters below 20 and 100 nm, on six human cell lines chosen as representative of potential target organs: HepG2 and Hep3B (liver), Caki-1 and Hek-293 (kidney), HT29 (intestine) and A549 (lung). Cytotoxicity of NDs was assessed by measuring cell impedance (xCELLigence® system) and cell survival/death by flow cytometry while genotoxicity was assessed by γ-H2Ax foci detection, which is considered the most sensitive technique for studying DNA double-strand breaks. To validate and check the sensitivity of the techniques, aminated polystyrene nanobeads were used as positive control in all assays. Cell incorporation of NDs was also studied by flow cytometry and luminescent N-V center photoluminescence (confirmed by Raman microscopy), to ensure that nanoparticles entered the cells. Overall, we show that NDs effectively entered the cells but NDs do not induce any significant cytotoxic or genotoxic effects on the six cell lines up to an exposure dose of 250 µg/mL. Taken together these results strongly support the huge potential of NDs for human nanomedicine but also their potential as negative control in nanotoxicology studies.

Advantages of flow cytometry immunophenotyping for the diagnosis of central nervous system non-Hodgkin's lymphoma in AIDS patients.

Science.gov (United States)

Subirá, D; Górgolas, M; Castañón, S; Serrano, C; Román, A; Rivas, F; Tomás, J F

2005-01-01

Neurological disorders are common in HIV-infected patients. Central nervous system (CNS) lymphoma should always be considered because it is an important cause of morbidity and mortality. To investigate the clinical utility of flow cytometry immunophenotyping (FCI) in diagnosing or discarding leptomeningeal involvement in HIV-infected patients and to compare its sensitivity with that of conventional cytological methods. Fifty-six cerebrospinal fluid (CSF) samples from 29 HIV-infected patients were independently evaluated by flow cytometry and cytology. The description of an aberrant immunophenotype was the criterion used to define the malignant nature of any CSF cell population. FCI and cytology gave concordant results for 48 of the 56 CSF samples studied: 37 were negative for malignancy and 11 had evidence of CNS lymphoma. Discordant results were obtained for eight CSF samples, and the accuracy of the FCI findings could be demonstrated for four CSF samples described as positive for malignancy according to the FCI criteria. A high level of agreement was found between the results obtained using the two methods, but FCI gave at least 25% higher sensitivity than conventional cytomorphological methods for the detection of malignant cells. This advantage suggests that, in case of negative flow cytometry results, disorders other than non-Hodgkin's lymphoma should be strongly considered.

Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements.

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Guérin, Frédéric; Arnaiz, Olivier; Boggetto, Nicole; Denby Wilkes, Cyril; Meyer, Eric; Sperling, Linda; Duharcourt, Sandra

2017-04-26

DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61

Imaging Flow Cytometry Analysis to Identify Differences of Survival Motor Neuron Protein Expression in Patients With Spinal Muscular Atrophy.

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Arakawa, Reiko; Arakawa, Masayuki; Kaneko, Kaori; Otsuki, Noriko; Aoki, Ryoko; Saito, Kayoko

2016-08-01

Spinal muscular atrophy is a neurodegenerative disorder caused by the deficient expression of survival motor neuron protein in motor neurons. A major goal of disease-modifying therapy is to increase survival motor neuron expression. Changes in survival motor neuron protein expression can be monitored via peripheral blood cells in patients; therefore we tested the sensitivity and utility of imaging flow cytometry for this purpose. After the immortalization of peripheral blood lymphocytes from a human healthy control subject and two patients with spinal muscular atrophy type 1 with two and three copies of SMN2 gene, respectively, we used imaging flow cytometry analysis to identify significant differences in survival motor neuron expression. A bright detail intensity analysis was used to investigate differences in the cellular localization of survival motor neuron protein. Survival motor neuron expression was significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. Moreover, survival motor neuron expression correlated with the clinical severity of spinal muscular atrophy according to SMN2 copy number. The cellular accumulation of survival motor neuron protein was also significantly decreased in cells derived from patients with spinal muscular atrophy relative to those derived from a healthy control subject. The benefits of imaging flow cytometry for peripheral blood analysis include its capacities for analyzing heterogeneous cell populations; visualizing cell morphology; and evaluating the accumulation, localization, and expression of a target protein. Imaging flow cytometry analysis should be implemented in future studies to optimize its application as a tool for spinal muscular atrophy clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

A New Series of Pyrrole-Based Chalcones: Synthesis and Evaluation of Antimicrobial Activity, Cytotoxicity, and Genotoxicity

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Ahmet Özdemir

2017-11-01

Full Text Available In an effort to develop new potent antimicrobial and anticancer agents, new pyrrole-based chalcones were designed and synthesized via the base-catalyzed Claisen-Schmidt condensation of 2-acetyl-1-methylpyrrole with 5-(arylfurfural derivatives. The compounds were evaluated for their in vitro antimicrobial effects on pathogenic bacteria and Candida species using microdilution and ATP luminescence microbial cell viability assays. MTT assay was performed to determine the cytotoxic effects of the compounds on A549 human lung adenocarcinoma, HepG2 human hepatocellular carcinoma, C6 rat glioma, and NIH/3T3 mouse embryonic fibroblast cell lines. 1-(1-Methyl-1H-pyrrol-2-yl-3-(5-(4-chlorophenylfuran-2-ylprop-2-en-1-one (7 and 1-(1-methyl-1H-pyrrol-2-yl-3-(5-(2,5-dichlorophenylfuran-2-ylprop-2-en-1-one (9 were found to be the most potent antifungal agents against Candida krusei and therefore these compounds were chosen for flow cytometry analysis and Ames MPF assay. ATP bioluminescence assay indicated that the antifungal activity of compounds 7 and 9 against C. krusei was significantly higher than that of other compounds and the reference drug (ketoconazole, whereas flow cytometry analysis revealed that the percentage of dead cells treated with compound 7 was more than that treated with compound 9 and ketoconazole. According to Ames MPF assay, compounds 7 and 9 were found to be non-genotoxic against TA98 and TA100 with/without metabolic activation. MTT assay indicated that 1-(1-methyl-1H-pyrrol-2-yl-3-(5-(2-nitrophenylfuran-2-ylprop-2-en-1-one (3 showed more selective anticancer activity than cisplatin against the HepG2 cell line. On the other hand, 1-(1-methyl-1H-pyrrol-2-yl-3-(5-(4-nitrophenylfuran-2-ylprop-2-en-1-one (1 was found to be more effective and selective on the A549 cell line than cisplatin.

Two new polyploid species closely related to Alnus glutinosa in Europe and North Africa - An analysis based on morphometry, karyology, flow cytometry and microsatellites

Czech Academy of Sciences Publication Activity Database

Vít, Petr; Douda, Jan; Krak, Karol; Havrdová, Alena; Mandák, Bohumil

2017-01-01

Roč. 66, č. 3 (2017), s. 567-583 ISSN 0040-0262 R&D Projects: GA ČR(CZ) GAP504/11/0402 Institutional support: RVO:67985939 Keywords : Alnus glutinosa * flow cytometry * Balkan Peninsula Subject RIV: EF - Botanics OBOR OECD: Plant sciences, botany Impact factor: 2.447, year: 2016

Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

Science.gov (United States)

Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

2017-03-01

Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

Remarkable coexistence of multiple cytotypes of the Gymnadenia conopsea aggregate (the fragrant orchid): evidence from flow cytometry

Czech Academy of Sciences Publication Activity Database

Trávníček, Pavel; Kubátová, B.; Čurn, V.; Rauchová, Jana; Krajníková, E.; Jersáková, J.; Suda, Jan

2011-01-01

Roč. 107, č. 1 (2011), s. 77-87 ISSN 0305-7364 Institutional research plan: CEZ:AV0Z6005908 Keywords : coexistence * contact zone * flow cytometry Subject RIV: EF - Botanics Impact factor: 4.030, year: 2011

Correlation between flow cytometry and histologic findings: ten year experience in the investigation of lymphoproliferative diseases

Directory of Open Access Journals (Sweden)

Alanna Mara Pinheiro Sobreira Bezerra

2011-06-01

Full Text Available Objective: To demonstrate the advantages of correlatingflow cytometry immunophenotyping with the pathology/immunohistochemistry of lymph nodes or nodules in the diagnosisof lymphoproliferative diseases. Methods: A retrospective studywas carried out of 157 biopsy or fine-needle aspiration lymph nodes/nodule specimens taken from 142 patients, from 1999 and 2009.The specimens were simultaneously studied with flow cytometryand pathology at Hospital Israelita Albert Einstein. The specimenswere prepared in hematoxylin/eosin, Giemsa, or monoclonal antibodystained slides for detecting specific antibodies for the purposesof pathology/immunohistochemical analysis. The samples werehemolyzed and marked with different monoclonal antibody panels fordifferent antigens in flow cytometry immunophenotyping. Results:The diagnostic results of pathology/immunohistochemical studiesand flow cytometry immunophenotyping agreed in 115 patients(81%, corresponding to 127 specimens, as follows according tothe pathologic diagnosis: 63 patients with non-Hodgkin’s B-celllymphoma; 26 patients with reactive lymphoid hyperplasia; 5 patientswith non-Hodgkin’s T-cell lymphoma; 4 patients with atypical lymphoidproliferation; 5 patients with a chronic granulomatous inflammatoryprocess; 5 patients with a non-hematologic diagnosis; 2 patientswith granulocytic sarcoma; 2 patients with thymoma; 1 patientwith byphenotypic leukemia; 1 patient with kappa plasmocytoma;1 patient with Hodgkin’s lymphoma. Subtypes of lymphomas couldbe classified by associating the two techniques: 19 patients withfollicular lymphoma; 15 patients with diffuse large B-cell lymphoma; 7patients with small lymphocytic B-cell lymphoma/chronic lymphocyticleukemia; 3 patients with mantle cell lymphoma; 1 patient withBurkitt’s lymphoma; 1 patient with MALT type lymphoma; 1 patientwith post-transplant lymphoproliferative disease; 2 patients with highgrade non-Hodgkin’s B-cell lymphoma; 1 patient with low grade

Analysis of cytotoxic effects of chlorhexidine gluconate as antiseptic agent on human blood lymphocytes.

Science.gov (United States)

Salimi, Ahmad; Alami, Bahare; Pourahmad, Jalal

2017-08-01

The aim of this study was to assess the cytotoxicity of chlorhexidine gluconate (CHG) on human blood lymphocytes as a useful ex vivo model for accelerated human toxicity studies. Using biochemical and flow cytometry assessments, we demonstrated that addition of CHG at 1 μM concentration to human blood lymphocytes induced cytotoxicity following 6 h. The CHG-induced cytotoxicity on human blood lymphocytes was associated with intracellular reactive oxygen species generation, mitochondrial membrane potential collapse, lysosomal membrane injury, lipid peroxidation, and depletion of glutathione. According to our results, CHG triggers oxidative stress and organelles damages in lymphocytes which are important cells in defense against foreign agents. Finally our findings suggest that using of antioxidants and mitochondrial/lysosomal protective agents could be of benefit for the people in the exposure with CHG. © 2017 Wiley Periodicals, Inc.

Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics

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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

Imaging flow cytometry assays for quantifying pigment grade titanium dioxide particle internalization and interactions with immune cells in whole blood.

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Hewitt, Rachel E; Vis, Bradley; Pele, Laetitia C; Faria, Nuno; Powell, Jonathan J

2017-10-01

Pigment grade titanium dioxide is composed of sub-micron sized particles, including a nanofraction, and is widely utilized in food, cosmetic, pharmaceutical, and biomedical industries. Oral exposure to pigment grade titanium dioxide results in at least some material entering the circulation in humans, although subsequent interactions with blood immune cells are unknown. Pigment grade titanium dioxide is employed for its strong light scattering properties, and this work exploited that attribute to determine whether single cell-particle associations could be determined in immune cells of human whole blood at "real life" concentrations. In vitro assays, initially using isolated peripheral blood mononuclear cells, identified titanium dioxide associated with the surface of, and within, immune cells by darkfield reflectance in imaging flow cytometry. This was confirmed at the population level by side scatter measurements using conventional flow cytometry. Next, it was demonstrated that imaging flow cytometry could quantify titanium dioxide particle-bearing cells, within the immune cell populations of fresh whole blood, down to titanium dioxide levels of 10 parts per billion, which is in the range anticipated for human blood following titanium dioxide ingestion. Moreover, surface association and internal localization of titanium dioxide particles could be discriminated in the assays. Overall, results showed that in addition to the anticipated activity of blood monocytes internalizing titanium dioxide particles, neutrophil internalization and cell membrane adhesion also occurred, the latter for both phagocytic and nonphagocytic cell types. What happens in vivo and whether this contributes to activation of one or more of these different cells types in blood merits further attention. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

flowAI: automatic and interactive anomaly discerning tools for flow cytometry data.

Science.gov (United States)

Monaco, Gianni; Chen, Hao; Poidinger, Michael; Chen, Jinmiao; de Magalhães, João Pedro; Larbi, Anis

2016-08-15

Flow cytometry (FCM) is widely used in both clinical and basic research to characterize cell phenotypes and functions. The latest FCM instruments analyze up to 20 markers of individual cells, producing high-dimensional data. This requires the use of the latest clustering and dimensionality reduction techniques to automatically segregate cell sub-populations in an unbiased manner. However, automated analyses may lead to false discoveries due to inter-sample differences in quality and properties. We present an R package, flowAI, containing two methods to clean FCM files from unwanted events: (i) an automatic method that adopts algorithms for the detection of anomalies and (ii) an interactive method with a graphical user interface implemented into an R shiny application. The general approach behind the two methods consists of three key steps to check and remove suspected anomalies that derive from (i) abrupt changes in the flow rate, (ii) instability of signal acquisition and (iii) outliers in the lower limit and margin events in the upper limit of the dynamic range. For each file analyzed our software generates a summary of the quality assessment from the aforementioned steps. The software presented is an intuitive solution seeking to improve the results not only of manual but also and in particular of automatic analysis on FCM data. R source code available through Bioconductor: http://bioconductor.org/packages/flowAI/ CONTACTS: mongianni1@gmail.com or Anis_Larbi@immunol.a-star.edu.sg Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Distinction between asymptomatic monoclonal B-cell lymphocytosis with cyclin D1 overexpression and mantle cell lymphoma: from molecular profiling to flow cytometry.

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Espinet, Blanca; Ferrer, Ana; Bellosillo, Beatriz; Nonell, Lara; Salar, Antonio; Fernández-Rodríguez, Concepción; Puigdecanet, Eulàlia; Gimeno, Javier; Garcia-Garcia, Mar; Vela, Maria Carmen; Luño, Elisa; Collado, Rosa; Navarro, José Tomás; de la Banda, Esmeralda; Abrisqueta, Pau; Arenillas, Leonor; Serrano, Cristina; Lloreta, Josep; Miñana, Belén; Cerutti, Andrea; Florensa, Lourdes; Orfao, Alberto; Sanz, Ferran; Solé, Francesc; Dominguez-Sola, David; Serrano, Sergio

2014-02-15

According to current diagnostic criteria, mantle cell lymphoma (MCL) encompasses the usual, aggressive variants and rare, nonnodal cases with monoclonal asymptomatic lymphocytosis, cyclin D1-positive (MALD1). We aimed to understand the biology behind this clinical heterogeneity and to identify markers for adequate identification of MALD1 cases. We compared 17 typical MCL cases with a homogeneous group of 13 untreated MALD1 cases (median follow-up, 71 months). We conducted gene expression profiling with functional analysis in five MCL and five MALD1. Results were validated in 12 MCL and 8 MALD1 additional cases by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in 24 MCL and 13 MALD1 cases by flow cytometry. Classification and regression trees strategy was used to generate an algorithm based on CD38 and CD200 expression by flow cytometry. We found 171 differentially expressed genes with enrichment of neoplastic behavior and cell proliferation signatures in MCL. Conversely, MALD1 was enriched in gene sets related to immune activation and inflammatory responses. CD38 and CD200 were differentially expressed between MCL and MALD1 and confirmed by flow cytometry (median CD38, 89% vs. 14%; median CD200, 0% vs. 24%, respectively). Assessment of both proteins allowed classifying 85% (11 of 13) of MALD1 cases whereas 15% remained unclassified. SOX11 expression by qRT-PCR was significantly different between MCL and MALD1 groups but did not improve the classification. We show for the first time that MALD1, in contrast to MCL, is characterized by immune activation and driven by inflammatory cues. Assessment of CD38/CD200 by flow cytometry is useful to distinguish most cases of MALD1 from MCL in the clinical setting. MALD1 should be identified and segregated from the current MCL category to avoid overdiagnosis and unnecessary treatment. ©2013 AACR

Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus.

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Jarzembowski, T; Wiśniewska, K; Józwik, A; Bryl, E; Witkowski, J

2008-08-01

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.

Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

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Werner, James H. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Larson, Erica J. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Goodwin, Peter M. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Ambrose, W. Patrick [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States); Keller, Richard A. [Division of Bioscience, Los Alamos National Laboratory, Mail Stop M888, Los Alamos, New Mexico 87545-0001 (United States)

2000-06-01

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America.

Effects of fluorescence excitation geometry on the accuracy of DNA fragment sizing by flow cytometry

International Nuclear Information System (INIS)

Werner, James H.; Larson, Erica J.; Goodwin, Peter M.; Ambrose, W. Patrick; Keller, Richard A.

2000-01-01

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual strained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the strained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution. (c) 2000 Optical Society of America

Flow Cytometry of the Side Population: Tips & Tricks

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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.

Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

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Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.

Identification and Characterization of Plasma Cells in Normal Human Bone Marrow by High-Resolution Flow Cytometry

NARCIS (Netherlands)

Terstappen, Leonardus Wendelinus Mathias Marie; Johnsen, Steen; Segers-Nolten, Gezina 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

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

Science.gov (United States)

Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

2013-07-25

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia.

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Wood, Brent L; Arroz, Maria; Barnett, David; DiGiuseppe, Joseph; Greig, Bruce; Kussick, Steven J; Oldaker, Teri; Shenkin, Mark; Stone, Elizabeth; Wallace, Paul

2007-01-01

Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. Copyright 2007 Clinical Cytometry Society.

Tyrosine kinase inhibitors as modulators of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity in breast cancer cell lines.

Science.gov (United States)

Collins, Denis M; Gately, Kathy; Hughes, Clare; Edwards, Connla; Davies, Anthony; Madden, Stephen F; O'Byrne, Kenneth J; O'Donovan, Norma; Crown, John

2017-09-01

Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6±4.7% (T12) by LDH assay and 19±3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. In the presence of effector cells with high

Characterization of glycosylphosphatidylinositol biosynthesis defects by clinical features, flow cytometry, and automated image analysis

DEFF Research Database (Denmark)

Knaus, Alexej; Pantel, Jean Tori; Pendziwiat, Manuela

2018-01-01

, the increasing number of individuals with a GPIBD shows that hyperphosphatasia is a variable feature that is not ideal for a clinical classification. METHODS: We studied the discriminatory power of multiple GPI-linked substrates that were assessed by flow cytometry in blood cells and fibroblasts of 39 and 14...... those with PIGA mutations. Although the impairment of GPI-linked substrates is supposed to play the key role in the pathophysiology of GPIBDs, we could not observe gene-specific profiles for flow cytometric markers or a correlation between their cell surface levels and the severity of the phenotype...

Bleaching response of Symbiodinium (zooxanthellae): determination by flow cytometry.

Science.gov (United States)

Lee, Co Sin; Yeo, Yin Sheng Wilson; Sin, Tsai Min

2012-10-01

Coral bleaching is of increasing concern to reef management and stakeholders. Thus far, quantification of coral bleaching tends to be heavily reliant on the enumeration of zooxanthellae, with less emphasis on assessment of photosynthetic or physiological condition, these being often assessed separately by techniques such as liquid chromatography. Traditional methods of enumeration using microscopy are time consuming, subjected to low precision and great observer error. In this study, we presented a method for the distinction of physoiological condition and rapid enumeration of zooxanthellae using flow cytometry (FCM). Microscopy verified that healthy looking/live versus damaged/dead zooxanthellae could be reliably and objectively distinguished and counted by FCM on the basis of red and green fluorescence and light scatter. Excellent correlations were also determined between FCM and microscopy estimates of cell concentrations of fresh zooxanthellae isolates from Pocillopora damicornis. The relative intensities of chlorophyll and β-carotene fluorescences were shown to be important in understanding the results of increased cell counts in freshly isolated zooxanthellae experimentally exposed to high temperatures (34, 36, and 38°C) over 24 h, with ambient temperature (29°C) used as controls. The ability to simultaneously identify and enumerate subpopulations of different physiological states in the same sample provides an enormous advantage in not just determining bleaching responses, but elucidating adaptive response and mechanisms for tolerance. Therefore, this approach might provide a rapid, convenient, and reproducible methodology for climate change studies and reef management programs. Copyright © 2012 International Society for Advancement of Cytometry.

Feasibility study of stain-free classification of cell apoptosis based on diffraction imaging flow cytometry and supervised machine learning techniques.

Science.gov (United States)

Feng, Jingwen; Feng, Tong; Yang, Chengwen; Wang, Wei; Sa, Yu; Feng, Yuanming

2018-06-01

This study was to explore the feasibility of prediction and classification of cells in different stages of apoptosis with a stain-free method based on diffraction images and supervised machine learning. Apoptosis was induced in human chronic myelogenous leukemia K562 cells by cis-platinum (DDP). A newly developed technique of polarization diffraction imaging flow cytometry (p-DIFC) was performed to acquire diffraction images of the cells in three different statuses (viable, early apoptotic and late apoptotic/necrotic) after cell separation through fluorescence activated cell sorting with Annexin V-PE and SYTOX® Green double staining. The texture features of the diffraction images were extracted with in-house software based on the Gray-level co-occurrence matrix algorithm to generate datasets for cell classification with supervised machine learning method. Therefore, this new method has been verified in hydrogen peroxide induced apoptosis model of HL-60. Results show that accuracy of higher than 90% was achieved respectively in independent test datasets from each cell type based on logistic regression with ridge estimators, which indicated that p-DIFC system has a great potential in predicting and classifying cells in different stages of apoptosis.

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.

A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

Directory of Open Access Journals (Sweden)

Erika Hammarlund

Full Text Available Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar could not be measured by plaque assay (PA, focus-forming assay (FFA, or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6 and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine. Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

DEFF Research Database (Denmark)

Pedersen, Natasja Wulff; Chandran, P. Anoop; Qian, Yu

2017-01-01

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide...... automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore...... laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0...

KHYG-1, a model for the study of enhanced natural killer cell cytotoxicity.

Science.gov (United States)

Suck, Garnet; Branch, Donald R; Smyth, Mark J; Miller, Richard G; Vergidis, Joanna; Fahim, Soad; Keating, Armand

2005-10-01

To compare the cytotoxicity of KHYG-1 with other natural killer (NK)/NK T-cell lines and identify molecules that may be associated with enhanced cytotoxicity, thereby eventually leading to improved NK cell-mediated cancer immunotherapy. NK/NK T-cell lines KHYG-1, NK-92, YT, and SNT-8 were compared with a novel flow cytometric cytotoxicity assay under different culture conditions. Transcription, expression, and phosphorylation studies were performed using polymerase chain reaction sequence-specific primers, reverse transcription polymerase chain reaction, immunoblotting, and flow cytometry. KHYG-1 is a highly cytotoxic cell line, exceeding the cytolytic capacity of the other cell lines against K562. KHYG-1 is also highly cytotoxic against the leukemia cell lines EM2, EM3, and HL60. The novel activation receptor NKp44 and its adaptor, DAP12, NKG2D, and constitutively phosphorylated ERK2 may be associated with the enhanced cytotoxicity of KHYG-1. This cell line most likely mediates cytolysis by granzyme M (but not granzymes A and B) together with perforin, which is constitutively fully cleaved to the 60-kD form, in contrast to the other cell lines. KHYG-1 is a valuable model for the study of enhanced cytotoxicity by NK cells. In addition to the activation of NKp44, KHYG-1 may induce apoptosis of tumor cells by the newly described granzyme M/perforin pathway. Targeted modifications of effector molecules demonstrated in this model could generate NK cells with even greater killing ability that may be particularly attractive for clinical application. Moreover, our demonstration of greater cytotoxicity of KHYG-1 versus NK-92 cells, already in clinical trials, suggests a direct therapeutic role for KHYG-1.

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

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.

Mapping cell populations in flow cytometry data for cross‐sample comparison using the Friedman–Rafsky test statistic as a distance measure

Science.gov (United States)

Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu

2015-01-01

Abstract Flow cytometry (FCM) is a fluorescence‐based single‐cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap‐FR, a novel method for cell population mapping across FCM samples. FlowMap‐FR is based on the Friedman–Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap‐FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap‐FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap‐FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap‐FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap‐FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback–Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL‐distance in distinguishing

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

Science.gov (United States)

Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Monitoring Immune Responses in Organ Recipients by Flow Cytometry

Directory of Open Access Journals (Sweden)

Al-Mukhalafi Zuha

2001-01-01

Full Text Available Allograft rejection remains a major barrier to successful organ transplan-tation. Cellular and humoral immune responses play a critical role in mediating graft rejection. During the last few years, monoclonal antibodies have been used as a new specific therapeutic approach in the prevention of allograft rejection. Recently, the technology of flow cytometry has become a useful tool for monitoring immunological responses in transplant recipients. The application of this valuable tool in clinical transplantation at the present time is aimed at, i determining the extent of immuno-suppressive therapy through T-cell receptor analysis of cellular components, ii monitoring levels of alloreactive antibodies to identify high-risk recipients (sensitized patients in the pre-operative period and iii to predict rejection by monitoring their development post-operatively. In future, further development of this technology may demonstrate greater benefit to the field of organ transplantation.

Increased Peripheral Blood Pro-Inflammatory/Cytotoxic Lymphocytes in Children with Bronchiectasis.

Directory of Open Access Journals (Sweden)

G Hodge

Full Text Available Bronchiectasis (BE in children is common in some communities including Indigenous children in Australia. Relatively little is known about the nature of systemic inflammation in these children, especially the contribution of specific pro-inflammatory and cytotoxic lymphocyte subsets: T-cells, natural killer (NK cells and NKT-like cells. We have shown that these cells produce increased cytotoxic (granzyme b and perforin and inflammatory (IFNγ and TNFα mediators in several adult chronic lung diseases and hypothesised that similar changes would be evident in children with BE.Intracellular cytotoxic mediators perforin and granzyme b and pro-inflammatory cytokines were measured in T cell subsets, NKT-like and NK cells from blood and bronchoalveolar samples from 12 children with BE and 10 aged-matched control children using flow cytometry.There was a significant increase in the percentage of CD8+ T cells and T and NKT-like subsets expressing perforin/granzyme and IFNγ and TNFα in blood in BE compared with controls. There was a further increase in the percentage of pro-inflammatory cytotoxic T cells in Indigenous compared with non-Indigenous children. There was no change in any of these mediators in BAL.Childhood bronchiectasis is associated with increased systemic pro-inflammatory/cytotoxic lymphocytes in the peripheral blood. Future studies need to examine the extent to which elevated levels of pro-inflammatory cytotoxic cells predict future co-morbidities.

Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

Directory of Open Access Journals (Sweden)

Hitoshi Uchiyama

2014-09-01

Full Text Available The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF. Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated. In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated. However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated. These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

Development of an unbiased, semi-automated approach for classifying plasma cell immunophenotype following multicolor flow cytometry of bone marrow aspirates.

Science.gov (United States)

Post, Steven R; Post, Ginell R; Nikolic, Dejan; Owens, Rebecca; Insuasti-Beltran, Giovanni

2018-03-24

Despite increased usage of multiparameter flow cytometry (MFC) to assess diagnosis, prognosis, and therapeutic efficacy (minimal residual disease, MRD) in plasma cell neoplasms (PCNs), standardization of methodology and data analysis is suboptimal. We investigated the utility of using the mean and median fluorescence intensities (FI) obtained from MFC to objectively describe parameters that distinguish plasma cell (PC) phenotypes. In this retrospective study, flow cytometry results from bone marrow aspirate specimens from 570 patients referred to the Myeloma Institute at UAMS were evaluated. Mean and median FI data were obtained from 8-color MFC of non-neoplastic, malignant, and mixed PC populations using antibodies to CD38, CD138, CD19, CD20, CD27, CD45, CD56, and CD81. Of 570 cases, 252 cases showed only non-neoplastic PCs, 168 showed only malignant PCs, and 150 showed mixed PC populations. Statistical analysis of median FI data for each CD marker showed no difference in expression intensity on non-neoplastic and malignant PCs, between pure and mixed PC populations. ROC analysis of the median FI of CD expression in non-neoplastic and malignant PCs was used to develop an algorithm to convert quantitative FI values to qualitative assessments including "negative," "positive," "dim," and "heterogeneous" expression. FI data derived from 8-color MFC can be used to define marker expression on PCs. Translation of FI data from Infinicyt software to an Excel worksheet streamlines workflow and eliminates transcriptional errors when generating flow reports. © 2018 International Clinical Cytometry Society. © 2018 International Clinical Cytometry Society.

Flow cytometry: design, development and experimental validation

International Nuclear Information System (INIS)

Seigneur, Alain

1987-01-01

The flow cytometry techniques allow the analysis and sorting of living biologic cells at rates above five to ten thousand events per second. After a short review, we present in this report the design and development of a 'high-tech' apparatus intended for research laboratories and the experimental results. The first part deals with the physical principles allowing morphologic and functional analysis of cells or cellular components. The measured parameters are as follows: electrical resistance pulse sizing, light scattering and fluorescence. Hydrodynamic centering is used, and in the same way, the division of a water-stream into droplets leading to electrostatic sorting of particles. The second part deals with the apparatus designed by the 'Commissariat a l'Energie Atomique' (C.E.A.) and industrialised by 'ODAM' (ATC 3000). The last part of this thesis work is the performance evaluations of this cyto-meter. The difference between the two size measurement methods are analyzed: electrical resistance pulse sizing versus small-angle light scattering. By an original optics design, high sensitivity has been reached in the fluorescence measurement: the equivalent noise corresponds to six hundred fluorescein isothiocyanate (FITC) molecules. The sorting performances have also been analyzed and the cell viability proven. (author) [fr

Microfluidics and photonics for Bio-System-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip.

Science.gov (United States)

Godin, Jessica; Chen, Chun-Hao; Cho, Sung Hwan; Qiao, Wen; Tsai, Frank; Lo, Yu-Hwa

2008-10-01

Microfluidics and photonics come together to form a field commonly referred to as 'optofluidics'. Flow cytometry provides the field with a technology base from which both microfluidic and photonic components be developed and integrated into a useful device. This article reviews some of the more recent developments to familiarize a reader with the current state of the technologies and also highlights the requirements of the device and how researchers are working to meet these needs.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Science.gov (United States)

Suzuki, Miho; Sakata, Ichiro; Sakai, Takafumi; Tomioka, Hiroaki; Nishigaki, Koichi; Tramier, Marc; Coppey-Moisan, Maïté

2015-12-15

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

An introduction to mass cytometry: fundamentals and applications.

Science.gov (United States)

Tanner, Scott D; Baranov, Vladimir I; Ornatsky, Olga I; Bandura, Dmitry R; George, Thaddeus C

2013-05-01

Mass cytometry addresses the analytical challenges of polychromatic flow cytometry by using metal atoms as tags rather than fluorophores and atomic mass spectrometry as the detector rather than photon optics. The many available enriched stable isotopes of the transition elements can provide up to 100 distinguishable reporting tags, which can be measured simultaneously because of the essential independence of detection provided by the mass spectrometer. We discuss the adaptation of traditional inductively coupled plasma mass spectrometry to cytometry applications. We focus on the generation of cytometry-compatible data and on approaches to unsupervised multivariate clustering analysis. Finally, we provide a high-level review of some recent benchmark reports that highlight the potential for massively multi-parameter mass cytometry.

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.

Multiparameter flow cytometry reveals myelodysplasia-related aberrant antigen expression in myelodysplastic/myeloproliferative neoplasms.

Science.gov (United States)

Kern, Wolfgang; Bacher, Ulrike; Schnittger, Susanne; Alpermann, Tamara; Haferlach, Claudia; Haferlach, Torsten

2013-05-01

Within the myelodysplastic/myeloproliferative neoplasm (MDS/MPN) category of the WHO (2008), only chronic myelomonocytic leukemia was so far evaluated by multiparameter flow cytometry (MFC). To investigate the potential of MFC for MDS/MPNs, unclassifiable (MDS/MPNu), and refractory anemia associated with ring sideroblasts and marked thrombocytosis (RARS-T), we studied 91 patients with these entities (60 males/31 females; 35.3-87.4 years) for MDS-related aberrant immunophenotypes (≥ 2 different cell lineages with ≥ 3 aberrantly expressed antigens). Data were correlated with cytomorphology and cytogenetics. MFC identified MDS-related immunophenotypes in 54/91 (59.3%) of patients. Patients with or without MDS-related immunophenotype did not differ significantly by demographic characteristics, blood values, or median overall survival. MDS-related immunophenotype cases showed a higher number of aberrantly expressed antigens (mean ± SD, 4.9 ± 2.4 vs. 2.0 ± 1.4; P MPNu and RARS-T. MFC therefore may be helpful to separate cases into more "MDS-like" or "MPN-like" subgroups. Copyright © 2012 International Clinical Cytometry Society.

Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland.

Science.gov (United States)

Glier, Hana; Heijnen, Ingmar; Hauwel, Mathieu; Dirks, Jan; Quarroz, Stéphane; Lehmann, Thomas; Rovo, Alicia; Arn, Kornelius; Matthes, Thomas; Hogan, Cassandra; Keller, Peter; Dudkiewicz, Ewa; Stüssi, Georg; Fernandez, Paula

2017-07-29

The EuroFlow Consortium developed a fully standardized flow cytometric approach from instrument settings, through antibody panel, reagents and sample preparation protocols, to data acquisition and analysis. The Swiss Cytometry Society (SCS) promoted a study to evaluate the feasibility of using such standardized measurements of 8-color data across two different flow cytometry platforms - Becton Dickinson (BD) FACSCanto II and Beckman Coulter (BC) Navios, aiming at increasing reproducibility and inter-laboratory comparability of immunophenotypic data in clinical laboratories in Switzerland. The study was performed in two phases, i.e. a learning phase (round 1) and an analytical phase (rounds 2 and 3) consisting of a total of three rounds. Overall, 10 laboratories using BD FACSCanto II (n=6) or BC Navios (n=4) flow cytometers participated. Each laboratory measured peripheral blood samples from healthy donors stained with a uniform antibody panel of reagents - EuroFlow Lymphoid Screening Tube (LST) - applying the EuroFlow standardized protocols for instrument setup and sample preparation (www.EuroFlow.org). All data files were analyzed centrally and median fluorescence intensity (MedFI) values for individual markers on defined lymphocyte subsets were recorded; variability from reference MedFI values was assessed using performance scores. Data troubleshooting and discussion of the results with the participants followed after each round at SCS meetings. The results of the learning phase demonstrated that standardized instrument setup and data acquisition are feasible in routine clinical laboratories without previous experience with EuroFlow. During the analytical phase, highly comparable data were obtained at the different laboratories using either BD FACSCanto II or BC Navios. The coefficient of variation of MedFI for 7 of 11 markers performed repeatedly below 30%. In the last study round, 89% of participants scored over 90% MedFI values within the acceptance criteria

Flow cytometry, microsatellites and niche models reveal the origins and geographical structure of Alnus glutinosa populations in Europe

Czech Academy of Sciences Publication Activity Database

Mandák, Bohumil; Vít, Petr; Krak, Karol; Trávníček, Pavel; Havrdová, Alena; Hadincová, Věroslava; Zákravský, Petr; Jarolímová, Vlasta; Bacles, C. F. E.; Douda, Jan

2016-01-01

Roč. 117, č. 1 (2016), s. 107-120 ISSN 0305-7364 R&D Projects: GA ČR(CZ) GAP504/11/0402 Institutional support: RVO:67985939 Keywords : flow cytometry * microsatellites * glacial refugia Subject RIV: EF - Botanics Impact factor: 4.041, year: 2016

B-ALL minimal residual disease flow cytometry: an application of a novel method for optimization of a single-tube model.

Science.gov (United States)

Shaver, Aaron C; Greig, Bruce W; Mosse, Claudio A; Seegmiller, Adam C

2015-05-01

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel. Copyright© by the American Society for Clinical Pathology.

Flow cytometry evidence of human granulocytes interaction with polyhedral oligomeric silsesquioxanes: effect of nanoparticle charge

International Nuclear Information System (INIS)

Renò, Filippo; Rizzi, Manuela; Pittarella, Pamela; Carniato, Fabio; Olivero, Francesco; Marchese, Leonardo

2013-01-01

Nanoparticles (NPs) entering the human body are immediately confronted with the innate part of human immune system. In particular, monocyte and neutrophil granulocytes readily clear particles by phagocytosis, even if in the case of NPs the uptake mechanism may be classified as macropinocytosis. Among engineered nanoparticles, in the last years, siliceous materials have emerged as promising materials for several applications ranging from catalysis to biomedical. The polyhedral oligomeric silsesquioxanes (POSS) are nanodimensional, easily synthesizable molecular compounds and POSS-based systems are promising carriers for biological molecules. In this work, the ability of human granulocytes to uptake positively and negatively charged POSS was measured using a simple flow cytometry analysis based on cell size modifications. The data obtained showed that after a 30 min exposure only positive NPs were uptaken by human granulocyte using both macropinocytosis and clathrin-mediated mechanisms as demonstrated by uptake inhibition mediated by amiloride and chlorpromazine. (paper)

DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

Science.gov (United States)

Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

2018-04-17

Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and

ggCyto: Next Generation Open-Source Visualization Software for Cytometry.

Science.gov (United States)

Van, Phu; Jiang, Wenxin; Gottardo, Raphael; Finak, Greg

2018-06-01

Open source software for computational cytometry has gained in popularity over the past few years. Efforts such as FlowCAP, the Lyoplate and Euroflow projects have highlighted the importance of efforts to standardize both experimental and computational aspects of cytometry data analysis. The R/BioConductor platform hosts the largest collection of open source cytometry software covering all aspects of data analysis and providing infrastructure to represent and analyze cytometry data with all relevant experimental, gating, and cell population annotations enabling fully reproducible data analysis. Data visualization frameworks to support this infrastructure have lagged behind. ggCyto is a new open-source BioConductor software package for cytometry data visualization built on ggplot2 that enables ggplot-like functionality with the core BioConductor flow cytometry data structures. Amongst its features are the ability to transform data and axes on-the-fly using cytometry-specific transformations, plot faceting by experimental meta-data variables, and partial matching of channel, marker and cell populations names to the contents of the BioConductor cytometry data structures. We demonstrate the salient features of the package using publicly available cytometry data with complete reproducible examples in a supplementary material vignette. https://bioconductor.org/packages/devel/bioc/html/ggcyto.html. gfinak@fredhutch.org. Supplementary data are available at Bioinformatics online and at http://rglab.org/ggcyto/.

The Diagnosis of Gastric Mucosa-associated Lymphoid Tissue Lymphoma by Flow Cytometry and Fluorescence in situ Hybridization of Biopsy Specimens.

Science.gov (United States)

Matsueda, Katsunori; Omote, Sizuma; Sakata, Masahiro; Fujita, Isao; Horii, Jouichiro; Toyokawa, Tatsuya

2018-04-15

Mucosa-associated lymphoid tissue (MALT) lymphoma and reactive inflammatory lymphoid changes are frequently difficult to distinguish based on a routine histological differential diagnosis. We were unable to diagnose gastric MALT lymphoma histologically using specimens obtained by endoscopy, although a flow cytometry (FCM) analysis demonstrated clonality of neoplastic cells by separating cells by CD45 gating. Furthermore, a fluorescence in situ hybridization (FISH) analysis showed trisomy 18. We therefore diagnosed gastric MALT lymphoma with trisomy 18. We recommend that FCM and FISH analyses of biopsy specimens be considered for diagnosing gastric MALT lymphoma if this diagnosis is suspected based on endoscopic findings.

Effectiveness of pulse-shape criteria for the selection of dicentric chromosomes by slit-scan flow cytometry and sorting

NARCIS (Netherlands)

Rens, W.; van Oven, C. H.; Stap, J.; Aten, J. A.

1993-01-01

A method was developed to detect dicentric chromosomes by slit-scan flow cytometry. The two centromeres of dicentric chromosomes are represented by the two dips in the trimodal fluorescence profile. A trimodal profile can, however, also be generated by aggregates of chromosomes. We tested the

Multi-channel imaging cytometry with a single detector

Science.gov (United States)

Locknar, Sarah; Barton, John; Entwistle, Mark; Carver, Gary; Johnson, Robert

2018-02-01

Multi-channel microscopy and multi-channel flow cytometry generate high bit data streams. Multiple channels (both spectral and spatial) are important in diagnosing diseased tissue and identifying individual cells. Omega Optical has developed techniques for mapping multiple channels into the time domain for detection by a single high gain, high bandwidth detector. This approach is based on pulsed laser excitation and a serial array of optical fibers coated with spectral reflectors such that up to 15 wavelength bins are sequentially detected by a single-element detector within 2.5 μs. Our multichannel microscopy system uses firmware running on dedicated DSP and FPGA chips to synchronize the laser, scanning mirrors, and sampling clock. The signals are digitized by an NI board into 14 bits at 60MHz - allowing for 232 by 174 pixel fields in up to 15 channels with 10x over sampling. Our multi-channel imaging cytometry design adds channels for forward scattering and back scattering to the fluorescence spectral channels. All channels are detected within the 2.5 μs - which is compatible with fast cytometry. Going forward, we plan to digitize at 16 bits with an A-toD chip attached to a custom board. Processing these digital signals in custom firmware would allow an on-board graphics processing unit to display imaging flow cytometry data over configurable scanning line lengths. The scatter channels can be used to trigger data buffering when a cell is present in the beam. This approach enables a low cost mechanically robust imaging cytometer.

Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice.

Science.gov (United States)

Liu, Liping; Yin, Yan; Li, Fei; Malhotra, Charvi; Cheng, Jianguo

2017-06-01

Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain. Copyright © 2017 Elsevier B.V. All rights reserved.

CMOS based image cytometry for detection of phytoplankton in ballast water.

Science.gov (United States)

Pérez, J M; Jofre, M; Martínez, P; Yáñez, M A; Catalan, V; Parker, A; Veldhuis, M; Pruneri, V

2017-02-01

We introduce an image cytometer (I-CYT) for the analysis of phytoplankton in fresh and marine water environments. A linear quantification of cell numbers was observed covering several orders of magnitude using cultures of Tetraselmis and Nannochloropsis measured by autofluorescence in a laboratory environment. We assessed the functionality of the system outside the laboratory by phytoplankton quantification of samples taken from a marine water environment (Dutch Wadden Sea, The Netherlands) and a fresh water environment (Lake Ijssel, The Netherlands). The I-CYT was also employed to study the effects of two ballast water treatment systems (BWTS), based on chlorine electrolysis and UV sterilization, with the analysis including the vitality of the phytoplankton. For comparative study and benchmarking of the I-CYT, a standard flow cytometer was used. Our results prove a limit of detection (LOD) of 10 cells/ml with an accuracy between 0.7 and 0.5 log, and a correlation of 88.29% in quantification and 96.21% in vitality, with respect to the flow cytometry results.

Optofluidic fluorescent imaging cytometry on a cell phone.

Science.gov (United States)

Zhu, Hongying; Mavandadi, Sam; Coskun, Ahmet F; Yaglidere, Oguzhan; Ozcan, Aydogan

2011-09-01

Fluorescent microscopy and flow cytometry are widely used tools in biomedical sciences. Cost-effective translation of these technologies to remote and resource-limited environments could create new opportunities especially for telemedicine applications. Toward this direction, here we demonstrate the integration of imaging cytometry and fluorescent microscopy on a cell phone using a compact, lightweight, and cost-effective optofluidic attachment. In this cell-phone-based optofluidic imaging cytometry platform, fluorescently labeled particles or cells of interest are continuously delivered to our imaging volume through a disposable microfluidic channel that is positioned above the existing camera unit of the cell phone. The same microfluidic device also acts as a multilayered optofluidic waveguide and efficiently guides our excitation light, which is butt-coupled from the side facets of our microfluidic channel using inexpensive light-emitting diodes. Since the excitation of the sample volume occurs through guided waves that propagate perpendicular to the detection path, our cell-phone camera can record fluorescent movies of the specimens as they are flowing through the microchannel. The digital frames of these fluorescent movies are then rapidly processed to quantify the count and the density of the labeled particles/cells within the target solution of interest. We tested the performance of our cell-phone-based imaging cytometer by measuring the density of white blood cells in human blood samples, which provided a decent match to a commercially available hematology analyzer. We further characterized the imaging quality of the same platform to demonstrate a spatial resolution of ~2 μm. This cell-phone-enabled optofluidic imaging flow cytometer could especially be useful for rapid and sensitive imaging of bodily fluids for conducting various cell counts (e.g., toward monitoring of HIV+ patients) or rare cell analysis as well as for screening of water quality in

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Choi, Seon Young; Jang, Soo Hwa [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of); Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su [Soongsil University, Department of Chemistry (Korea, Republic of); Lee, Kangtaek [Yonsei University, Department of Chemical and Biomolecular Engineering (Korea, Republic of); Yang, Sung Ik [Kyung Hee University, College of Environment and Applied Chemistry (Korea, Republic of); Joo, Sang-Woo, E-mail: sjoo@ssu.ac.kr [Soongsil University, Department of Chemistry (Korea, Republic of); Ryu, Pan Dong; Lee, So Yeong, E-mail: leeso@snu.ac.kr [Seoul National University, Laboratory of Veterinary Pharmacology, College of Veterinary Medicine and Institute for Veterinary Science (Korea, Republic of)

2012-12-15

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Cellular uptake and cytotoxicity of positively charged chitosan gold nanoparticles in human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Choi, Seon Young; Jang, Soo Hwa; Park, Jin; Jeong, Saeromi; Park, Jin Ho; Ock, Kwang Su; Lee, Kangtaek; Yang, Sung Ik; Joo, Sang-Woo; Ryu, Pan Dong; Lee, So Yeong

2012-01-01

Cellular uptake, cytotoxicity, and mechanisms of cytotoxicity of the positively charged Au nanoparticles (NPs) were examined in A549 cells, which are one of the most characterized pulmonary cellular systems. Positively charged Au NPs were prepared by chemical reduction using chitosan. The dimension and surface charge of Au NPs were examined by transmission electron microscopy (TEM), dynamic light scattering, and zeta potential measurements. The uptake of Au NPs into A549 cells was also monitored using TEM and dark-field microscopy (DFM) and z-stack confocal microRaman spectroscopy. DFM live cell imaging was also performed to monitor the entry of chitosan Au NPs in real time. The cytotoxic assay, using both methylthiazol tetrazolium and lactate dehydrogenase assays revealed that positively charged Au NPs decreased cell viability. Flow cytometry, DNA fragmentation, real-time PCR, and western blot analysis suggest that positively charged chitosan Au NPs provoke cell damage through both apoptotic and necrotic pathways.

Assessment of a flow cytometry technique for studying signaling pathways in platelets: Monitoring of VASP phosphorylation in clinical samples

Directory of Open Access Journals (Sweden)

N. Mallouk

2018-07-01

Full Text Available A recently released kit (PerFix EXPOSE was reported to improve the measurement of the degree of phosphorylation of proteins in leukocytes by flow cytometry. We tested its adaptation for platelets to monitor vasodilator-stimulated phosphoprotein (VASP phosphorylation, which is the basis of a currently used test for the assessment of the pharmacological response to P2Y12 antagonists (PLT VASP/P2Y12. The PerFix EXPOSE kit was compared to the PLT VASP/P2Y12 kit by using blood samples drawn at 24 h post clopidogrel dose from 19 patients hospitalized for a non-cardio-embolic ischemic stroke and treated with clopidogrel monotherapy for at least five days in an observational study. The platelet PerFix method was based on adaptation of the volume of the sample, the centrifugation speed and the incubation temperature. Poor agreement between prevention by adenosine diphosphate (ADP of PGE1-induced cAMP-mediated VASP phosphorylation and ADP induced aggregation assessed by Light Transmittance Aggregometry was found. We found a significant correlation between the PLT VASP/P2Y12 kit and the PerFix EXPOSE kit. The PerFix EXPOSE kit may also be helpful to monitor adverse effects of second-generation tyrosine kinase inhibitors on platelets. Keywords: Platelet signaling, VASP, Flow cytometry, Clopidogrel

Preferentially Cytotoxic Constituents of Andrographis paniculata and their Preferential Cytotoxicity against Human Pancreatic Cancer Cell Lines.

Science.gov (United States)

Lee, Sullim; Morita, Hiroyuki; Tezuka, Yasuhiro

2015-07-01

In the course of our search for anticancer agents based on a novel anti-austerity strategy, we found that the 70% EtOH extract of the crude drug Andrographis Herba (aerial parts of Andrographis paniculata), used in Japanese Kampo medicines, killed PANC-1 human pancreatic cancer cells preferentially in nutrient-deprived medium (NDM). Phytochemical investigation of the 70% EtOH extract led to the isolation of 21 known compounds consisting of six labdane-type diterpenes (11, 15, 17-19, 21), six flavones (5, 7, 10, 12, 14, 20), three flavanones (2, 6, 16), two sterols (3, 8), a fatty acid (1), a phthalate (4), a triterpene (9), and a monoterpene (13). Among them, 14-deoxy-11,12-didehydroandrographolide (17) displayed the most potent preferential cytotoxicity against PANC-1 and PSN-1 cells with PC50 values of 10.0 μM and 9.27 μM, respectively. Microscopical observation, double staining with ethidium bromide (EB) and acridine orange (AO), and flow cytometry with propidium iodide/annexin V double staining indicated that 14-deoxy-11,12-didehydroandrographolide (17) triggered apoptosis-like cell death in NDM with an amino acids and/or serum-sensitive mode.

Immunological Tools: Engaging Students in the Use and Analysis of Flow Cytometry and Enzyme-linked Immunosorbent Assay (ELISA)

Science.gov (United States)

Ott, Laura E.; Carson, Susan

2014-01-01

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques associated with clinical and research applications within the immunology and medical fields. The use of these techniques is becoming increasingly valuable in many life science and engineering disciplines as well. Herein, we report the development and…

Coupling Bacterial Activity Measurements with Cell Sorting by Flow Cytometry.

Science.gov (United States)

Servais; Courties; Lebaron; Troussellier

1999-08-01

> Abstract A new procedure to investigate the relationship between bacterial cell size and activity at the cellular level has been developed; it is based on the coupling of radioactive labeling of bacterial cells and cell sorting by flow cytometry after SYTO 13 staining. Before sorting, bacterial cells were incubated in the presence of tritiated leucine using a procedure similar to that used for measuring bacterial production by leucine incorporation and then stained with SYTO 13. Subpopulations of bacterial cells were sorted according to their average right-angle light scatter (RALS) and fluorescence. Average RALS was shown to be significantly related to the average biovolume. Experiments were performed on samples collected at different times in a Mediterranean seawater mesocosm enriched with nitrogen and phosphorus. At four sampling times, bacteria were sorted in two subpopulations (cells smaller and larger than 0.25 µm(3)). The results indicate that, at each sampling time, the growth rate of larger cells was higher than that of smaller cells. In order to confirm this tendency, cell sorting was performed on six subpopulations differing in average biovolume during the mesocosm follow-up. A clear increase of the bacterial growth rates was observed with increasing cell size for the conditions met in this enriched mesocosm.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p180.html

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

NARCIS (Netherlands)

Dionisio Pires, L.M.; 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

Design and Application of Sensors for Chemical Cytometry.

Science.gov (United States)

Vickerman, Brianna M; Anttila, Matthew M; Petersen, Brae V; Allbritton, Nancy L; Lawrence, David S

2018-02-08

The bulk cell population response to a stimulus, be it a growth factor or a cytotoxic agent, neglects the cell-to-cell variability that can serve as a friend or as a foe in human biology. Biochemical variations among closely related cells furnish the basis for the adaptability of the immune system but also act as the root cause of resistance to chemotherapy by tumors. Consequently, the ability to probe for the presence of key biochemical variables at the single-cell level is now recognized to be of significant biological and biomedical impact. Chemical cytometry has emerged as an ultrasensitive single-cell platform with the flexibility to measure an array of cellular components, ranging from metabolite concentrations to enzyme activities. We briefly review the various chemical cytometry strategies, including recent advances in reporter design, probe and metabolite separation, and detection instrumentation. We also describe strategies for improving intracellular delivery, biochemical specificity, metabolic stability, and detection sensitivity of probes. Recent applications of these strategies to small molecules, lipids, proteins, and other analytes are discussed. Finally, we assess the current scope and limitations of chemical cytometry and discuss areas for future development to meet the needs of single-cell research.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

Science.gov (United States)

Sachdeva, Man Updesh Singh; Varma, Neelam; Chandra, Dinesh; Bose, Parveen; Malhotra, Pankaj; Varma, Subhash

2015-05-01

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Cell proliferation and apoptosis in the primary enamel knot measured by flow cytometry of laser microdissected samples

Czech Academy of Sciences Publication Activity Database

Matalová, Eva; Dubská, L.; Fleischmannová, Jana; Chlastáková, Ivana; Janečková, Eva; Tucker, A. S.

2010-01-01

Roč. 55, č. 8 (2010), s. 570-575 ISSN 0003-9969 R&D Projects: GA AV ČR KJB500450802; GA AV ČR IAA600450904; GA ČR GA203/08/1680 Institutional research plan: CEZ:AV0Z50450515 Keywords : Laser capture microdissection * Flow cytometry * Primary enamel knot Subject RIV: EA - Cell Biology Impact factor: 1.463, year: 2010

Flow Cytometry Detection of Infectious Rotaviruses in Environmental and Clinical Samples

Science.gov (United States)

Abad, F. Xavier; Pintó, Rosa M.; Bosch, Albert

1998-01-01

A method for the detection of infectious human rotaviruses based on infection of CaCo-2 cells and detection of infected cells by indirect immunofluorescence and flow cytometry (IIF-FC) has been developed. The technique was validated by performing a seminested reverse transcription-PCR assay with sorted cell populations. The efficiency of the procedure has been compared with that of the standard method of infection of MA104 cells and ulterior detection by IIF and optical microscopy (IIF-OM) and with that of infection of MA104 cells and detection by IIF-FC. The limit of sensitivity for the detection of the cell-adapted strain Itor P13, expressed as the most probable number of cytopathogenic units, was established as 200 and 2 for MA104 and CaCo-2 cells, respectively, by the IIF-FC method. The ratio of infectious virus particles to total virus particles for a wild-type rotavirus was determined to be 1/2 × 106 and 1/2 × 104 for IIF-OM with MA104 cells and IIF-FC with CaCo-2 cells, respectively. The use of IIF-FC with CaCo-2 cells was tested with fecal and water samples and proved to be more effective than the standard procedure for rotavirus detection. PMID:9647805

Suppression of natural killer cell cytotoxicity in postpartum women: time course and potential mechanisms.

Science.gov (United States)

Groer, Maureen W; El-Badri, Nagwa; Djeu, Julie; Williams, S Nicole; Kane, Bradley; Szekeres, Karoly

2014-07-01

Little is known about the recovery of the immune system from normal pregnancy and whether the postpartum period is a uniquely adapted immune state. This report extends previous observations from our group of decreased natural killer (NK) cell cytotoxicity in the postpartum period. NK cytotoxicity was measured from 1 week through 9 months postpartum. In addition, NK cytotoxicity was assayed in the presence or absence of pooled plasmas collected from either postpartum or nonpostpartum women. Samples of cells were stained for inhibitory receptors and analyzed by flow cytometry. NK cytotoxicity remained decreased in postpartum women compared to controls through the first 6 postpartum months, returned to normal levels by 9 months, and remained normal at 12 months. NK cytotoxicity during the first 6 months was further inhibited by the addition of pooled plasma to NK cultures from postpartum women, but the addition of pooled plasma from the control group did not affect that group's NK cultures. There were differences in inhibitory receptor staining between the two groups, with decreased CD158a and CD158b and increased NKG2A expression on postpartum NK cells during the first 3 postpartum months. These data suggest that NK cytotoxicity postpartum inhibition lasts 6 months and is influenced by unidentified postpartum plasma components. The effect may also involve receptors on NK cells. © The Author(s) 2013.

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.M.; Van Donk, E.; 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

Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

International Nuclear Information System (INIS)

Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H.; Santelli, Glaucia M.M.

2017-01-01

Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

Flow cytometry based micronucleus assay for evaluation of genotoxic potential of 2-ACBs in hepatic cells HepG2

Energy Technology Data Exchange (ETDEWEB)

Barbezan, Angélica B.; Santos, Carla J.B.; Carvalho, Luma R.; Vieira, Daniel P.; Villavicêncio, Anna L.C.H., E-mail: abarbezan@ipen.br [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), São Paulo, SP (Brazil); Santelli, Glaucia M.M. [Universidade de São Paulo (USP), SP (Brazil). Departamento de Biologia Celular e do Desenvolvimento

2017-07-01

Food irradiation is approved for use in more than 60 countries for applications and purposes in a wide variety of foods, being an effective and safe method for preservation and long-term storage. 2-Alkylcyclobutanones (2-ACBs) are the only known radiolytic products generated from foods that contain fatty acids (Triglycerides) when irradiated. The acids analyzed in this study are palmitic and stearic, which when irradiated form 2-Dodecylcyclobutanones (2-dDCB) and 2-Tetradecylcyclobutanone (2-tDCB). Part of the 2-ACBs ingested is excreted through feces and part is deposited in adipose tissues. In vitro studies so far have been only in colon cells. The work used a human hepatoma cell line (HepG2) since the accumulation of fat in this organ is quite common. Micronucleus test was selected to evaluate possible genotoxic effects of 2-dDCB and 2-tDCB compounds when exposed to high concentrations (447, 1422 and 2235 μM) for 4 and 24 hours. Tests were performed in quadriplicates using flow cytometric analysis. None detectable genotoxic damage was observed after 4 hours of exposure to the compounds, and cytotoxic effects were only significant at the highest concentration (2235 μM) of 2-dDCB. After 24 hours of exposure, slight genotoxic damage was observed at all concentrations evaluated, and cytotoxic effects were only present when exposed to compound 2-tDCB. Although there is a genotoxic and cytotoxic effect in some of the situations tested, the two compounds predominantly induced proliferation reduction effects of this hepatic tumor cell line. (author)

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 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. Furthermore, this method can provide additional information in case of disagreement between in vivo and in vitro test results.

Ploidy Levels among Species in the ‘Oxalis tuberosa Alliance’ as Inferred by Flow Cytometry

OpenAIRE

EMSHWILLER, EVE

2002-01-01

The ‘Oxalis tuberosa alliance’ is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as ‘oca’. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C‐values in the alliance by estimating nuclear DNA contents of these acc...

Effect of acetic acid on Saccharomyces carlsbergensis ATCC 6269 batch ethanol production monitored by flow cytometry.

Science.gov (United States)

Freitas, Cláudia; Neves, Elisabete; Reis, Alberto; Passarinho, Paula C; da Silva, Teresa Lopes

2012-11-01

Bioethanol produced from lignocellulosic materials has been considered a sustainable alternative fuel. Such type of raw materials have a huge potential, but their hydrolysis into mono-sugars releases toxic compounds such as weak acids, which affect the microorganisms' physiology, inhibiting the growth and ethanol production. Acetic acid (HAc) is the most abundant weak acid in the lignocellulosic materials hydrolysates. In order to understand the physiological changes of Saccharomyces carlsbergensis when fermenting in the presence of different acetic acid (HAc) concentrations, the yeast growth was monitored by multi-parameter flow cytometry at same time that the ethanol production was assessed. The membrane potential stain DiOC(6)(3) fluorescence intensity decreased as the HAc concentration increased, which was attributed to the plasmic membrane potential reduction as a result of the toxic effect of the HAc undissociated form. Nevertheless, the proportion of cells with permeabilized membrane did not increase with the HAc concentration increase. Fermentations ending at lower external pH and higher ethanol concentrations depicted the highest proportions of permeabilized cells and cells with increased reactive oxygen species levels. Flow cytometry allowed monitoring, near real time (at-line), the physiological states of the yeast during the fermentations. The information obtained can be used to optimize culture conditions to improve bioethanol production.

An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

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Jenner, Dominic; Chong, Damien; Walker, Nicola; Green, A Christopher

2018-02-01

The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

Altered effector function of peripheral cytotoxic cells in COPD

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Corne Jonathan M

2009-06-01

Full Text Available Abstract Background There is mounting evidence that perforin and granzymes are important mediators in the lung destruction seen in COPD. We investigated the characteristics of the three main perforin and granzyme containing peripheral cells, namely CD8+ T lymphocytes, natural killer (NK; CD56+CD3- cells and NKT-like (CD56+CD3+ cells. Methods Peripheral blood mononuclear cells (PBMCs were isolated and cell numbers and intracellular granzyme B and perforin were analysed by flow cytometry. Immunomagnetically selected CD8+ T lymphocytes, NK (CD56+CD3- and NKT-like (CD56+CD3+ cells were used in an LDH release assay to determine cytotoxicity and cytotoxic mechanisms were investigated by blocking perforin and granzyme B with relevant antibodies. Results The proportion of peripheral blood NKT-like (CD56+CD3+ cells in smokers with COPD (COPD subjects was significantly lower (0.6% than in healthy smokers (smokers (2.8%, p +CD3- cells from COPD subjects were significantly less cytotoxic than in smokers (16.8% vs 51.9% specific lysis, p +CD3+ cells (16.7% vs 52.4% specific lysis, p +CD3- and NKT-like (CD56+CD3+ cells from smokers and HNS. Conclusion In this study, we show that the relative numbers of peripheral blood NK (CD56+CD3- and NKT-like (CD56+CD3+ cells in COPD subjects are reduced and that their cytotoxic effector function is defective.

Flow cytometry for rapid detection of Salmonella spp. in seed sprouts

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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.

Detection of effect cytotoxic of the alpha hemolysin of E. Coli (HLY A) in leukocytes polymorphonuclear neutrophils by means of cytometry of flow

International Nuclear Information System (INIS)

Garcia, J.

2000-01-01

Cell viability of Hly A exposed polymorphonuclear neutrophils (PMN) was assessed by propidium uptake, measured by flow cytometry. Hemolytic supernatant, but not the non hemolytic controls, caused a dose-dependent fluorescence signal in PMN. Cells exposed to low hemolytic activities (bellow 0.5 HU50/ml) did not fluoresce, although cell size, estimate by Forward Scatter (FSC), increased slightly, and returned to normal within 30-60 minutes suggesting both membrane damage in absence of propodium uptake and term cell recovery from the effects of Hly A. The fluorescent signal from permeated PMN decrease 15 minutes after exposure to Hly a, a decrease which was prevented by chelation ok extracellular Ca +2 with EGTA. Whereas Ca +2 entry into the cell is responsible for triggering mechanisms leading to loss of fluorescence, low or chelated extracelular Ca +2 facilitate propidium uptake, but the fluorescent signal does not decrease only when both intracellular and extracellular Ca +2 are chelated. The findings of this study, together whit data from other authors, are taken as basis to formulate a hypothetical sequence of events to explain the cytometric data obtained from Hly A exposed PMN, including the significance of increases in cell size without propidium uptake. (Author) [es

MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian Systems.

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Hanna L Sladitschek

Full Text Available The continuous improvement of imaging technologies has driven the development of sophisticated reporters to monitor biological processes. Such constructs should ideally be assembled in a flexible enough way to allow for their optimization. Here we describe a highly reliable cloning method to efficiently assemble constructs for imaging or flow cytometry applications in mammalian cell culture systems. We bioinformatically identified a list of restriction enzymes whose sites are rarely found in human and mouse cDNA libraries. From the best candidates, we chose an enzyme combination (MluI, XhoI and SalI: MXS that enables iterative chaining of individual building blocks. The ligation scar resulting from the compatible XhoI- and SalI-sticky ends can be translated and hence enables easy in-frame cloning of coding sequences. The robustness of the MXS-chaining approach was validated by assembling constructs up to 20 kb long and comprising up to 34 individual building blocks. By assessing the success rate of 400 ligation reactions, we determined cloning efficiency to be 90% on average. Large polycistronic constructs for single-cell imaging or flow cytometry applications were generated to demonstrate the versatility of the MXS-chaining approach. We devised several constructs that fluorescently label subcellular structures, an adapted version of FUCCI (fluorescent, ubiquitination-based cell cycle indicator optimized to visualize cell cycle progression in mouse embryonic stem cells and an array of artificial promoters enabling dosage of doxycyline-inducible transgene expression. We made publicly available through the Addgene repository a comprehensive set of MXS-building blocks comprising custom vectors, a set of fluorescent proteins, constitutive promoters, polyadenylation signals, selection cassettes and tools for inducible gene expression. Finally, detailed guidelines describe how to chain together prebuilt MXS-building blocks and how to generate new

Autologous Pure Platelet-Rich Plasma Dermal Injections for Facial Skin Rejuvenation: Clinical, Instrumental, and Flow Cytometry Assessment.

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Cameli, Norma; Mariano, Maria; Cordone, Iole; Abril, Elva; Masi, Serena; Foddai, Maria Laura

2017-06-01

Platelet-rich plasma (PRP) is an emerging treatment in dermatology recently proposed for skin rejuvenation. To evaluate the efficacy and safety of autologous pure PRP dermal injections on facial skin rejuvenation, investigating the cellularity of PRP samples. Twelve patients underwent 3 sessions of PRP injection at 1-month intervals. The clinical and instrumental outcomes were evaluated before (T0) and 1 month (T1) after the end of treatment by means of transepidermal water loss, corneometry, Cutometer, Visioscan, and Visioface. A flow cytometry characterization on PRP and peripheral blood (PB) samples was performed. Clinical and patient evaluation showed improvement of skin texture. Skin gross elasticity, skin smoothness parameters, skin barrier function, and capacitance were significantly improved. No difference between PRP and PB lymphocyte immunological asset was observed. A leukocyte population (mainly CD3) and neutrophils depletion were documented in all the PRP samples. This instrumental study demonstrated that PRP poor in leukocytes can provide objective improvements in skin biostimulation. Flow cytometry showed no variability among the PRP samples using a reproducible separation system and a low content in proinflammatory cells. Although a pilot study, it may be helpful for future investigations on PRP cellularity.

Comparison of BALB/c and CBA/J mice for the local lymph node assay using bromodeoxyuridine with flow cytometry (LLNA: BrdU-FCM).

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Lee, Yong Sun; Yi, Jung-Sun; Seo, Souk Jin; Kim, Joo Hwan; Jung, Mi-Sook; Seo, Im-Kwon; Ahn, Ilyoung; Ko, Kyungyuk; Kim, Tae Sung; Lim, Kyung Min; Sohn, Soojung

2017-02-01

The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells (LNCs) with flow cytometry. Unlike other LLNA methods (OECD TG 429, 442A and 442B) in which the CBA/J mouse strain is used, LLNA: BrdU-FCM was originally designed to be compatible with BALB/c, a mouse strain that is more widely used in many countries. To justify the substitution of CBA/J for BALB/c, the equivalence of the test results between two strains shall be established prior to the official implementation of LLNA: BrdU-FCM. This study aims to compare the test results of LLNA: BrdU-FCM produced in BALB/c mice with those in CBA/J mice for 18 reference substances, including 13 sensitizers and 5 non-sensitizers, listed in OECD Test Guideline 429. Based on the LLNA: BrdU-FCM test procedure, we selected an appropriate solvent and then performed preliminary tests to determine the non-irritating dose ranges for the main study, which revealed the difference in the irritation responses to 8 of the 18 chemicals between the two strains. In the main study, we measured the changes in the number of total LNCs, which indicated differences in the responses to test chemicals between the two strains. However, the stimulation index obtained with the counts of BrdU-incorporated LNCs with 7-AAD using flow cytometry yielded comparable results and 100% concordance between the BALB/c and CBA/J mouse strains was achieved, suggesting that the performance of LLNA: BrdU-FCM using BALB/c mice was equivalent to that with CBA/J mice. Copyright © 2016 Elsevier Inc. All rights reserved.

A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

International Nuclear Information System (INIS)

Patibandla, Phani K; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

2014-01-01

Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (∼45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

Cytotoxic Properties of Three Isolated Coumarin-hemiterpene Ether Derivatives from Artemisia armeniaca Lam.

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Mojarrab, Mahdi; Emami, Seyed Ahmad; Delazar, Abbas; Tayarani-Najaran, Zahra

2017-01-01

Considering multiple reports on cytotoxic activity of the Artemisia genus and its phytochemicals, in the current study A. armeniaca Lam. and the three components isolated from the plant were subjected to cytotoxic studies. Analytical fractionation of A. armeniaca aerial parts for the first time was directed to the isolation of 7-hydroxy-8-(4-hydroxy-3-methylbutoxy) comarin (armenin), 8-hydroxy-7-(4-hydroxy-3-methylbutoxy) comarin (isoarmenin) and deoxylacarol. Cytotoxicity assessed with alamalBlue® assay and apoptosis was detected by PI staining and western blot analysis of Bax and PARP proteins. Extracts and all compounds exhibited cytotoxic activity against apoptosis-proficient HL-60 and apoptosis-resistant K562 cells, with the lowest cytotoxic activity on J774 cell line as non-malignant cell. Armenin as the most potent component decreased the viability of cell with IC50 of 22.5 and 71.1 µM for K562 and HL-60 cells respectively and selected for further mechanistic study. Armenin increased the sub-G1 peak in flow cytometry histogram of HL-60 and K562 treated cells and increase in the amount of Bax protein and the cleavage of PARP in comparison with the control after treatment for 48 h in K562 treated cells verified the apoptotic activity of the armenin. Taken together, according to the finding of this study armenin was introduced as a novel cytotoxic compound with apoptotic activity, which is encouraging for further mechanistic and clinical studies.

Microfabrication and Test of a Three-Dimensional Polymer Hydro-focusing Unit for Flow Cytometry Applications

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Yang, Ren; Feeback, Daniel L.; Wang, Wan-Jun

2005-01-01

This paper details a novel three-dimensional (3D) hydro-focusing micro cell sorter for micro flow cytometry applications. The unit was microfabricated by means of SU-8 3D lithography. The 3D microstructure for coaxial sheathing was designed, microfabricated, and tested. Three-dimensional hydrofocusing capability was demonstrated with an experiment to sort labeled tanned sheep erythrocytes (red blood cells). This polymer hydro-focusing microstructure is easily microfabricated and integrated with other polymer microfluidic structures. Keywords: SU-8, three-dimensional hydro-focusing, microfluidic, microchannel, cytometer

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

Kelussia odoratissima potentiates cytotoxic effects of radiation in HeLa cancer cell line

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Azar Hosseini

2017-02-01

Full Text Available Objective: Cervical cancer is the second most common cause of death from cancer in women throughout the world. The aim of this study was to evaluate the cytotoxic activity of Kelussia odoratissima (K. odoratissima extract associated with radiotherapy in cervical cancer cells (HeLa cell line.Materials and Methods: Different concentration of the extract (25-500µg/ml was tested in HeLa cell lines. Cell cytotoxicity of the extract and the effects of the extract on radiation (2Gy/min-induced damages were assessed by MTT assay. Apoptosis was assessed using flow cytometric analysis.Result: K. odoratissima decreased cell viability in HeLa cell line in a concentration and time-dependent manner. When compared to the control,K. odoratissima induced a sub-G1 peak in the flow cytometry histogram of treated cells, indicating that apoptotic cell death is involved in K. odoratissima-induced toxicity. It was also shown that K. odoratissima sensitizes cells to radiation-induced toxicity.Conclusion: Our result showed the extract increased the radiation effect. This observation may be related to the presence of active compounds such as phthalides and ferulic acid.

Use of a Whole-Cell Biosensor and Flow Cytometry to Detect AHL Production by an Indigenous Soil Community During Decomposition

DEFF Research Database (Denmark)

Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

2005-01-01

originating from Vibrio fischeri. This resulted in a whole-cell biosensor, responding to the presence of AHL compounds. The biosensor was introduced to compost soil microcosms amended with nettle leaves. After 3 days of incubation, cells were extracted and analyzed by flow cytometry. All microcosms contained...

Lack of dependence of 5-fluorodeoxyuridine-mediated radiosensitization on cytotoxicity

International Nuclear Information System (INIS)

Lawrence, T.S.; Davis, M.A.; Chang, E.Y.

1995-01-01

It has been proposed that fluoropyrimidine-mediated cytotoxicity and radiosensitization are closely correlated. We have shown that HT29 human colon cancer cells transfected with the E. coli dUTPase gene are resistant to 5-fluorodeoxyuridine (FdUrd)-mediated cytotoxicity, presumably through more effective elimination of dUTP. We used these cells to assess the association between radiosensitization and cytotoxicity produced by FdUrd. The radiation sensitivities of the clones expressing elevated dUTPase activity (dutE clones) were similar to those of untransfected HT29 cells or HT29 cells which has been transfected with only the expression vector for the E. coli gene (con clones). We found that FdUrd produced similar increases in radiation sensitivity regardless of dUTPase activity. Levels of dUTPase in the dutE clones remained elevated during the entire period of FdUrd exposure, demonstrating that the lack of difference between dutE and Con clones was not a reflection of down-regulation of dUTPase activity by FdUrd, Flow cytometry showed that all clones progressed past the G 1 /S-phase boundary and into early S phase during FdUrd treatment. These data suggest that the mechanisms of FdUrd-mediated cytotoxicity and radiosensitization are not closely linked. These findings, combined with our previous investigations, are consistent with the hypothesis that radiosensitization occurs in cells which progress past the G 1 /S-phase boundary in the presence of FdUrd. 24 refs., 2 figs., 2 tabs

Antibacterial and Cytotoxic Activity of Compounds Isolated from Flourensia oolepis

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Mariana Belén Joray

2015-01-01

Full Text Available The antibacterial and cytotoxic effects of metabolites isolated from an antibacterial extract of Flourensia oolepis were evaluated. Bioguided fractionation led to five flavonoids, identified as 2′,4′-dihydroxychalcone (1, isoliquiritigenin (2, pinocembrin (3, 7-hydroxyflavanone (4, and 7,4′-dihydroxy-3′-methoxyflavanone (5. Compound 1 showed the highest antibacterial effect, with minimum inhibitory concentration (MIC values ranging from 31 to 62 and 62 to 250 μg/mL, against Gram-positive and Gram-negative bacteria, respectively. On further assays, the cytotoxic effect of compounds 1–5 was determined by MTT assay on acute lymphoblastic leukemia (ALL and chronic myeloid leukemia (CML cell lines including their multidrug resistant (MDR phenotypes. Compound 1 induced a remarkable cytotoxic activity toward ALL cells (IC50 = 6.6–9.9 μM and a lower effect against CML cells (IC50 = 27.5–30.0 μM. Flow cytometry was used to analyze cell cycle distribution and cell death by PI-labeled cells and by Annexin V/PI staining, respectively. Upon treatment, 1 induced cell cycle arrest in the G2/M phase accompanied by a strong induction of apoptosis. These results describe for the first time the antibacterial metabolites of F. oolepis extract, with 1 being the most effective. This chalcone also emerges as a selective cytotoxic agent against sensitive and resistant leukemic cells, highlighting its potential as a lead compound.

New mononuclear leukocyte-like populations within the granulocyte scatter gate detected by flow cytometry (Conference Presentation)

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Melzer, Susanne; Löffler, Markus; Kautzner, Marlene; Tárnok, Attila

2017-02-01

Granulocytes are the major players in innate immunity and are prognostic markers in diseases. An in-depth phenotypic characterization of granulocyte subtypes and correlation with biometry or lifestyle is so far lacking. The reason is, that either preparation of mononuclear cells was analyzed or that cells in the neutrophil window were neglected in the analysis. Here we show for the first time lymphocyte- (LL) and monocyte-like (ML) cells within the granulocyte scatter gate as new, previously unknown cell subpopulation. Immunophenotyping of 905 healthy German adults from the LIFE study [1] was performed by 10-color flow cytometry [2]. Age of men (n=420): 56.5±14.0 years, women (n=485): 56.7±13.6 y (range of 18-81 y). Data analyzed by FlowJo v10.0.6. Values compared by Mann-Whitney-U test: men vs women, young (18-49 y) vs. elderly (50-81 y.) men, and young (19-49 y.) vs. elderly (50-81 y.) women; significance: page, except LL2 in women. In conclusion, new lymphocyte like cell types with the neutrophil scatter characteristics are reported. Counts correlate with age and gender. We plan to sort these new subtypes for further functional characterization and aim to establish them as cellular biomarkers for the early detection of various diseases. [1] BMC Public Health. 2015;15:691; [2] Cytometry A. 2014;85(9):781

Effects of acute gamma-irradiation on spermatogenesis as revealed by flow cytometry

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Hacker, U; Schumann, J; Goehde, W [Muenster Univ. (Germany, F.R.)

1980-01-01

Mice irradiated with doses ranging from 0.1 to 15 Gy using a /sup 60/Co-source and controls were killed at intervals varying from 2 to 35 days after irradiation. The DNA content of the testicular cells in single cell suspensions was measured with the pulse cytophotometer to determine the frequencies of the different stages in the spermatogenesis. The relative amount of S-phase and 4c-cells was reduced initially but increased subsequently to hypernormal values. A decrease of 2c-cells indicated a higher cell-kill of diploid spermatogonia. Gamma ray-induced spermatids with abnormal DNA-values (diploid sperm) were identified. The results suggest that the spermatogenesis can be analysed with flow cytometry and used as a biologic dosimeter even for small doses of ionizing radiation.

Effects of acute gamma-irradiation on spermatogenesis as revealed by flow cytometry

International Nuclear Information System (INIS)

Hacker, U.; Schumann, J.; Goehde, W.

1980-01-01

Mice irradiated with doses ranging from 0.1 to 15 Gy using a 60 Co-source and controls were killed at intervals varying from 2 to 35 days after irradiation. The DNA content of the testicular cells in single cell suspensions was measured with the pulse cytophotometer to determine the frequencies of the different stages in the spermatogenesis. The relative amount of S-phase and 4c-cells was reduced initially but increased subsequently to hypernormal values. A decrease of 2c-cells indicated a higher cell-kill of diploid spermatogonia. Gamma ray-induced spermatids with abnormal DNA-values (diploid sperm) were identified. The results suggest that the spermatogenesis can be analysed with flow cytometry and used as a biologic dosimeter even for small doses of ionizing radiation. (Auth.)

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status.

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D'Hondt, Liesbet; Höfte, Monica; Van Bockstaele, Erik; Leus, Leen

2011-10-01

Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

A comparative evaluation of Eosin-5'-maleimide flow cytometry reveals a high diagnostic efficacy for hereditary spherocytosis.

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Joshi, P; Aggarwal, A; Jamwal, M; Sachdeva, M U S; Bansal, D; Malhotra, P; Sharma, P; Das, R

2016-10-01

Laboratory diagnosis of hereditary spherocytosis (HS) relies on increased incubated red cell osmotic fragility test for screening. We evaluated the diagnostic role of eosin-5'-maleimide (EMA) binding test by flow cytometry in spherocytic and microcytic hypochromic hematological disorders in North Indians. EMA binding test using flow cytometry was performed on 55 HS (40 families), 26 iron deficiency anemia (IDA), 32 β-thalassemia trait (βTT), and 10 autoimmune hemolytic anemia (AIHA) cases and 121 normals. Mean channel fluorescence (MCF) and coefficient of variation (CV) were studied. Different MCF parameters (MCF, MCF ratio, percent decrease MCF) and percent increase in CV were analyzed. Receiver operating characteristics analysis was performed to determine best cutoff values, sensitivity, and specificity for discriminating HS from other red cell disorders. MCF ratio of HS group was significantly lower than normals (0.67 ± 0.07 vs. 1.01 ± 0.05, P < 0.001) and other cases. All patients with HS showed MCF ratio to be ≤0.79. Four postsplenectomy cases with near-normal hemograms also revealed low MCF ratio, showing the specificity of the test. EMA assay was efficient to diagnose cases of HS including postsplenectomy cases and shows no overlap with IDA, βTT, and AIHA. © 2016 John Wiley & Sons Ltd.

Evaluation of Escherichia coli viability by flow cytometry: A method for determining bacterial responses to antibiotic exposure.

Science.gov (United States)

Boi, Paola; Manti, Anita; Pianetti, Anna; Sabatini, Luigia; Sisti, Davide; Rocchi, Marco Bruno; Bruscolini, Francesca; Galluzzi, Luca; Papa, Stefano

2015-01-01

In this study, we check for the presence of specific resistance genes by polymerase chain reaction (PCR) and then we used flow cytometry (FCM) to evaluate antibiotic-induced effects in different strains of Escherichia coli (E. coli). The presence of resistance genes was investigated by PCR in 10 strains of E. coli isolated from Foglia River. Bacterial responses to different antibiotics were also tested with FCM techniques by evaluating both the degree of decrease in viability and the light scatter changes in all of the strains. PCR revealed that only one strain exhibits the presence of one resistance gene. Despite this, analyses of strains using FCM evidenced the presence of viable subpopulations after antibiotic treatment. Furthermore, analyses of scatter signals revealed profound changes in the Forward Scatter and Side Scatter of the bacterial populations as a consequence of antibiotic exposure, confirming the viability and membrane potential data. The riverine strains were in general less sensitive to antibiotics than the reference strain (ATCC 25922). Antibiotic resistance is a widespread phenomena. The multiparametric approach based on FCM used in this study, providing results about different aspects (cell viability, membrane potential, light scatter changes), may overcome the limitation of PCR and could represent an adequate method for the evaluation of bacteria responses to antibiotic exposure. © 2014 International Clinical Cytometry Society.

Epigenetic targeting in acute myeloid leukemia: use of flow cytometry in monitoring therapeutic effects.

Science.gov (United States)

Ryningen, Anita; Bruserud, Øystein

2007-12-01

Flow cytometric techniques have emerged as a powerful tool in hematology allowing fast, sensitive and reproducible multi-parametric analyses at the single cell level of heterogeneous samples. Small subsets of cells can be studied with high degree of accuracy, and a broad and constantly increasing specter of antibodies is available. Flow cytometry has therefore become the method of choice for evaluation of therapeutic effects at single cell level. These methodological approaches can easily be used to study hematological malignancies, and the future use of this strategy in other malignancies will depend on the development of laboratory techniques to prepare suspensions of viable cells also from tumor biopsies. The selection of biological parameters for evaluation of treatment effects should probably be based on (i) molecular markers involved in cancer-associated genetic abnormalities; (ii) other molecular markers showing altered expression in the malignant cells and thought to be involved in leukemogenesis or having a prognostic impact; (ii) functional assays known to reflect biological characteristics that are important in carcinogenesis (e.g. cell cycle distribution, functional evaluation of apoptosis regulation). These molecules will in addition often represent the therapeutic targets when new anticancer drugs are developed. In this review we use treatment of acute myeloid leukemia with histone deacetylase inhibitors as an example. Based on the criteria mentioned above we suggest that the monitoring of therapeutic effects on the cancer cells in these patients should include differentiation status, histone acetylation, cell cycle distribution, pro- and anti-apoptotic signaling balance and intracellular levels of various transcription factors.

Contribution of flow cytometry to the diagnosis of gastric lymphomas in endoscopic biopsy specimens.

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Almasri, N M; Zaer, F S; Iturraspe, J A; Braylan, R C

1997-07-01

Gastric lymphomas seem to have unique clinical, pathologic, and immunophenotypic features that set them apart from nodal lymphomas. Microscopic examination of endoscopic biopsy specimens is the most frequent procedure used to diagnose gastric tumors, but it is very difficult, and sometimes impossible, to recognize lymphomas in endoscopic samples by histologic or even immunohistologic methods. Because most gastric lymphomas are of B-cell origin, we used flow cytometry to assess B-cell clonality in gastric biopsy specimens containing dense lymphocytic infiltrates thought to represent lymphoma. We prepared viable cell suspensions from unfixed specimens obtained from 29 consecutive patients who had a previous microscopic diagnosis of suspicious gastric lymphoid infiltrates. We performed immunophenotypic studies with multicolor flow cytometry, and we assessed clonality by examination of immunoglobulin (Ig) light-chain expression analyzed exclusively on B cells identified by anti-CD20 or CD19 antibodies. The mean number of cells recovered was 1.04 x 10(6), from an average of 5.5 gastric biopsy fragments per patient. In 26 of the 29 patients, the number of cells was adequate for analysis. We detected B-cell monoclonality in 16 cases, including 5 in which the percentage of clonal B cells was less than 5%. Of the 16 cases, only 8 could be diagnosed as lymphomas on morphologic grounds alone; the remaining 8 patients had either suspicious lymphoid infiltrates or chronic gastritis. The three cases with an insufficient number of cells were considered non-neoplastic either on histologic grounds alone or in conjunction with Southern analysis of Ig genes. We conclude that flow cytometric immunophenotypic analysis of freshly prepared cell suspensions obtained from endoscopic biopsy specimens can be used to evaluate gastric lymphocytic infiltrates. Specifically, the analysis of surface Ig light-chain expression on B cells distinguishes between monoclonal (lymphoma) and polyclonal

Comprehensive Analysis of the Chemical Composition and In Vitro Cytotoxic Mechanisms of Pallines Spinosa Flower and Leaf Essential Oils Against Breast Cancer Cells

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Ayman M Saleh

2017-08-01

Full Text Available Background/Aims: In our quest for new natural anticancer agents, we studied the cytotoxicity of the essential oils extracted from flowers and leaves of Pallines spinosa. Methods: The essential oils were extracted by hydrodistillation and solid phase microextraction (SPME from flowers and leaves of the plant and their composition was determined by GC/GC-MS. The cytotoxicity of the oils was evaluated against MCF-7 and MDA-MB-231 breast adenocarcinomas, and the non-cancerous MCF-10-2A cells, using a flow cytometry-based assay Apoptosis was evaluated by flow cytometry, nuclear staining, caspases activation, and Western blotting techniques, and cell cycle by measuring DNA contents. Results: The hydrodistilled flower oil contained mainly sesquiterpenes (96.39%, while the leaf sample was dominated by oxygenated-sesquiterpenes (51.60% and sesquiterpene-hydrocarbons (34.06%. In contrast, the SPME oil contained mainly monoterpene-hydrocarbons (44.09% and sesquiterpene-hydrocarbons (34.15% in the flower and leaf samples, respectively. The cytotoxicity of the flower oil against MCF-7 (IC50 0.25 ± 0.03 µg/mL and MDA-MB-231 (IC50 0.21 ± 0.03 µg/mL was much stronger than the leaf oil (IC50 2.4 ± 0.5 µg/mL and 1.5 ± 0.1 µg/mL, respectively. The toxicity of the flower oil was ∼5 to 8-times less in normal MCF-10-2A (IC50 1.3 ± 0.2 µg/mL and blood mononuclear cells (2.80 ± 0.45 µg/mL as compared to breast and hematological cancer cells, respectively. Both oils induced a caspase-dependent and -independent apoptosis in MCF-7 and MDA-MB-231 cells, and altered the levels of Bcl-2 and Bax proteins. In addition, the oils arrested cell cycle in both cancer cell lines at G0/G1 phase by modulating the expression of cyclin D1, CDK4 and p21 proteins. Conclusion: The cytotoxicity of P. spinosa oils were mediated by apoptosis and cell cycle arrest, suggesting the potential use of their bioactive compounds as natural anticancer compounds.

A novel quantitative kinase assay using bacterial surface display and flow cytometry.

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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.

A rapid method for infectivity titration of Andes hantavirus using flow cytometry.

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Barriga, Gonzalo P; Martínez-Valdebenito, Constanza; Galeno, Héctor; Ferrés, Marcela; Lozach, Pierre-Yves; Tischler, Nicole D

2013-11-01

The focus assay is currently the most commonly used technique for hantavirus titer determination. This method requires an incubation time of between 5 and 11 days to allow the appearance of foci after several rounds of viral infection. The following work presents a rapid Andes virus (ANDV) titration assay, based on viral nucleocapsid protein (N) detection in infected cells by flow cytometry. To this end, an anti-N monoclonal antibody was used that was developed and characterized previously. ANDV N could be detected as early as 6 h post-infection, while viral release was not observed until 24-48 h post-infection. Given that ANDV detection was performed during its first round of infection, a time reduction for titer determination was possible and provided results in only two days. The viral titer was calculated from the percentage of N positive cells and agreed with focus assay titers. Furthermore, the assay was applied to quantify the inhibition of ANDV cell entry by patient sera and by preventing endosome acidification. This novel hantavirus titration assay is a highly quantitative and sensitive tool that facilitates infectivity titration of virus stocks, rapid screening for antiviral drugs, and may be further used to detect and quantify infectious virus in human samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid identification of Staphylococcus aureus and methicillin resistance by flow cytometry using a peptide nucleic acid probe.

Science.gov (United States)

Shrestha, Nabin K; Scalera, Nikole M; Wilson, Deborah A; Brehm-Stecher, Byron; Procop, Gary W

2011-09-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively.

A rapid, reliable method of evaluating growth and viability of intraerythrocytic protozoan hemoparasites using fluorescence flow cytometry

OpenAIRE

Davis,W. C.; Wyatt,C. R.; Hamilton,M. J.; Goff,W. L.

1992-01-01

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethiedine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrone, ethidium. Following uptake of the dye, erythrocytes contamine viable parasites were readily distinguished and quantitated. Timed s...

Use of flow cytometry to identify monoclonal antibodies that recognize conserved epitopes on orthologous leukocyte differentiation antigens in goats, llamas, and rabbits

Czech Academy of Sciences Publication Activity Database

Davis, W. C.; Drbal, Karel; El-Aziz, A.; Mosaad, A.E.; Elbagory, A.R.M.; TIbary, A.; Barrington, G.M.; Park, Y.H.; Hamilton, M.J.

2007-01-01

Roč. 119, 1-2 (2007), s. 123-130 ISSN 0165-2427 Institutional research plan: CEZ:AV0Z50520514 Keywords : flow cytometry * monoclonal antibodies * leukocytes Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.957, year: 2007

Detecting Lactococcus lactis Prophages by Mitomycin C-Mediated Induction Coupled to Flow Cytometry Analysis

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Joana Oliveira

2017-07-01

Full Text Available Most analyzed Lactococcus lactis strains are predicted to harbor one or more prophage genomes within their chromosome; however, the true extent of the inducibility and functionality of such prophages cannot easily be deduced from sequence analysis alone. Chemical treatment of lysogenic strains with Mitomycin C is known to cause induction of temperate phages, though it is not always easy to clearly identify a lysogenic strain or to measure the number of released phage particles. Here, we report the application of flow cytometry as a reliable tool for the detection and enumeration of released lactococcal prophages using the green dye SYTO-9.

Relationship of CD86 surface marker expression and cytotoxicity on dendritic cells exposed to chemical allergen

International Nuclear Information System (INIS)

Hulette, Ben C.; Ryan, Cindy A.; Gildea, Lucy A.; Gerberick, G. Frank

2005-01-01

Human peripheral blood-derived dendritic cells (DC) respond to a variety of chemical allergens by up-regulating expression of the co-stimulatory molecule CD86. It has been postulated that this measure might provide the basis for an in vitro alternative approach for the identification of skin sensitizing chemicals. We recently reported that DC, exposed in culture to the highest non-cytotoxic concentrations of various chemical allergens, displayed marginal up-regulation of membrane CD86 expression; the interpretation being that such changes were insufficiently sensitive for the purposes of hazard identification. For the work presented here, immature DC were derived from human monocytes and treated with the chemical allergens 2,4-dinitrobenzenesulfonic acid (DNBS), nickel sulfate (NiSO 4 ), p-phenylenediamine (PPD), Bandrowski's base (BB), hydroquinone (HQ) and propyl gallate (PG) for 48 h at concentrations which induced both no to slight to moderate cytotoxicity. For comparison, DC were treated with the irritants sodium dodecyl sulfate (SDS), benzoic acid (BA), and benzalkonium chloride (BZC) at concentrations resulting in comparable levels of cytotoxicity. CD86 expression, as measured by flow cytometry, was consistently up-regulated (ranging from 162 to 386% control) on DC treated with concentrations of chemical allergens that induced approximately 10-15% cytotoxicity. The irritants BA and BZC did not induce up-regulation of CD86 expression when tested at concentrations that induced similar levels of cytotoxicity. SDS, however, up-regulated CD86 expression to 125-138% of control in 2/4 preparations when tested at concentrations which induced similar toxicity. Our results confirm that chemical allergens up-regulate CD86 expression on blood-derived DC and illustrate further that up-regulation of CD86 surface marker expression is more robust when DC are treated with concentrations of chemical allergen that induce slight to moderate cytotoxicity

Prostate extracellular vesicles in patient plasma as a liquid biopsy platform for prostate cancer using nanoscale flow cytometry

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Al-Zahrani, Ali A.; Pardhan, Siddika; Brett, Sabine I.; Guo, Qiu Q.; Yang, Jun; Wolf, Philipp; Power, Nicholas E.; Durfee, Paul N.; MacMillan, Connor D.; Townson, Jason L.; Brinker, Jeffrey C.; Fleshner, Neil E.; Izawa, Jonathan I.; Chambers, Ann F.; Chin, Joseph L.; Leong, Hon S.

2016-01-01

Background Extracellular vesicles released by prostate cancer present in seminal fluid, urine, and blood may represent a non-invasive means to identify and prioritize patients with intermediate risk and high risk of prostate cancer. We hypothesize that enumeration of circulating prostate microparticles (PMPs), a type of extracellular vesicle (EV), can identify patients with Gleason Score≥4+4 prostate cancer (PCa) in a manner independent of PSA. Patients and Methods Plasmas from healthy volunteers, benign prostatic hyperplasia patients, and PCa patients with various Gleason score patterns were analyzed for PMPs. We used nanoscale flow cytometry to enumerate PMPs which were defined as submicron events (100-1000nm) immunoreactive to anti-PSMA mAb when compared to isotype control labeled samples. Levels of PMPs (counts/μL of plasma) were also compared to CellSearch CTC Subclasses in various PCa metastatic disease subtypes (treatment naïve, castration resistant prostate cancer) and in serially collected plasma sets from patients undergoing radical prostatectomy. Results PMP levels in plasma as enumerated by nanoscale flow cytometry are effective in distinguishing PCa patients with Gleason Score≥8 disease, a high-risk prognostic factor, from patients with Gleason Score≤7 PCa, which carries an intermediate risk of PCa recurrence. PMP levels were independent of PSA and significantly decreased after surgical resection of the prostate, demonstrating its prognostic potential for clinical follow-up. CTC subclasses did not decrease after prostatectomy and were not effective in distinguishing localized PCa patients from metastatic PCa patients. Conclusions PMP enumeration was able to identify patients with Gleason Score ≥8 PCa but not patients with Gleason Score 4+3 PCa, but offers greater confidence than CTC counts in identifying patients with metastatic prostate cancer. CTC Subclass analysis was also not effective for post-prostatectomy follow up and for

CD3+CD4negCD8neg (double negative) T lymphocytes and NKT cells as the main cytotoxic-related-CD107a+ cells in lesions of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.

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Ferraz, Raquel; Cunha, Clarissa F; Pimentel, Maria Inês F; Lyra, Marcelo R; Pereira-Da-Silva, Tatiana; Schubach, Armando O; Da-Cruz, Alda Maria; Bertho, Alvaro Luiz

2017-05-03

Cutaneous leishmaniasis (CL) is caused by Leishmania (Viannia) braziliensis, which infects dermal macrophages and dendritic cells, causing an intense immune-mediated-tissue inflammation and a skin ulcer with elevated borders that can heal spontaneously or after antimonial therapy. The resolution of lesions depends on an adaptive immune response, and cytotoxic cells seem to have a fundamental role in this process. The aim of this study is to better understand the role of cytotoxicity mediated mechanisms that occur during the immune response in the CL lesion milieu, considering distinct cytotoxic-related CD107a + cells, such as CD8 + , CD4 + , CD4 neg CD8 neg (double-negative, DN) and CD4 + CD8 + (double-positive, DP) T lymphocytes, as well as NK and NKT cells. Lesion derived cells were assessed for T cell subpopulations and NK cells, as well as CD107a expression by flow cytometry. In addition, cytometric bead array (CBA) was used to quantify cytokines and granzyme B concentrations in supernatants from macerated lesions. Flow cytometry analyses revealed that NKT cells are the major CD107a-expressing cell population committed to cytotoxicity in CL lesion, although we also observed high frequencies of CD4 + and DN T cells expressing CD107a. Analysing the pool of CD107a + -cell populations, we found a higher distribution of DN T cells (44%), followed by approximately 25% of NKT cells. Interestingly, NK and CD8 + T cells represented only 3 and 4% of the total-CD107a + -cell pool, respectively. The cytotoxicity activity that occurs in the lesion milieu of CL patients seems to be dominated by DN T and NKT cells. These findings suggest the need for a reevaluation of the role of classical-cytotoxic NK and CD8 + T cells in the pathogenesis of CL, implicating an important role for other T cell subpopulations.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

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Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

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McBee, Megan E; Chionh, Yok H; Sharaf, Mariam L; Ho, Peiying; Cai, Maggie W L; Dedon, Peter C

2017-01-01

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis , and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled

Cutting-edge analysis of extracellular microparticles using ImageStream(X) imaging flow cytometry.

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Headland, Sarah E; Jones, Hefin R; D'Sa, Adelina S V; Perretti, Mauro; Norling, Lucy V

2014-06-10

Interest in extracellular vesicle biology has exploded in the past decade, since these microstructures seem endowed with multiple roles, from blood coagulation to inter-cellular communication in pathophysiology. In order for microparticle research to evolve as a preclinical and clinical tool, accurate quantification of microparticle levels is a fundamental requirement, but their size and the complexity of sample fluids present major technical challenges. Flow cytometry is commonly used, but suffers from low sensitivity and accuracy. Use of Amnis ImageStream(X) Mk II imaging flow cytometer afforded accurate analysis of calibration beads ranging from 1 μm to 20 nm; and microparticles, which could be observed and quantified in whole blood, platelet-rich and platelet-free plasma and in leukocyte supernatants. Another advantage was the minimal sample preparation and volume required. Use of this high throughput analyzer allowed simultaneous phenotypic definition of the parent cells and offspring microparticles along with real time microparticle generation kinetics. With the current paucity of reliable techniques for the analysis of microparticles, we propose that the ImageStream(X) could be used effectively to advance this scientific field.

Use of flow cytometry for the adhesion analysis of Streptococcus pyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase and cysteine protease, and the transcriptional regulator Rgg

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Finne Jukka

2006-02-01

Full Text Available Abstract Background Flow cytometry based adherence assay is a potentially powerful but little used method in the study of bacterial binding to host structures. We have previously characterized a glycoprotein-binding activity in Streptococcus pyogenes called 'strepadhesin' binding to thyroglobulin, submaxillar mucin, fetuin and asialofetuin. We have identified surface-associated pullulanase (PulA and cysteine protease (SpeB as carriers of strepadhesin activity. In the present paper, we investigated the use of flow cytometry as a method to study the binding of Rgg, SpeB and PulA knock-out strains to cultured human epithelial cells. Results Streptococcal mutants were readily labelled with CFDA-SE and their binding to epithelial cells could be effectively studied by flow cytometry. A strain deficient in Rgg expression showed increased binding to the analyzed epithelial cell lines of various origin. Inactivation of SpeB had no effect on the adhesion, while PulA knock-out strains displayed decreased binding to the cell lines. Conclusion These results suggest that the flow cytometric assay is a valuable tool in the analysis of S. pyogenes adherence to host cells. It appears to be an efficient and sensitive tool for the characterization of interactions between the bacteria and the host at the molecular level. The results also suggest a role for Rgg regulated surface molecules, like PulA, in the adhesion of S. pyogenes to host cells.

Magnetic microgels for drug targeting applications: Physical–chemical properties and cytotoxicity evaluation

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Turcu, Rodica, E-mail: rodica.turcu@itim-cj.ro [National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath Street, 400293 Cluj-Napoca (Romania); Craciunescu, Izabell [National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath Street, 400293 Cluj-Napoca (Romania); Garamus, Vasil M. [Helmholtz-Zentrum Geesthacht, Zentrum für Material- und Küstenforschung GmbH, 21502 Geesthacht (Germany); Janko, Christina; Lyer, Stefan; Tietze, Rainer; Alexiou, Christoph [ENT-Department, Else Kröner-Fresenius Stiftung-Professorship, Section for Experimental Oncology and Nanomedicine (SEON), University Hospital Erlangen (Germany); Vekas, Ladislau, E-mail: vekas@acad-tim.tm.edu.ro [Romanian Academy-Timisoara Branch, CFATR, Laboratory of Magnetic Fluids, Mihai Viteazul Street 24, 300223 Timisoara (Romania)

2015-04-15

Magnetoresponsive microgels with high saturation magnetization values have been obtained by a strategy based on the miniemulsion method using high colloidal stability organic carrier ferrofluid as primary material. Hydrophobic nanoparticles Fe{sub 3}O{sub 4}/oleic acid are densely packed into well-defined spherical nanoparticle clusters coated with polymers with sizes in the range 40–350 nm. Physical–chemical characteristics of magnetic microgels were investigated by TEM, SAXS, XPS and VSM measurements with the focus on the structure–properties relationship. The impact of magnetic microgels loaded with anticancer drug mitoxantrone (MTO) on the non-adherent human T cell leukemia line Jurkat was investigated in multiparameter flow cytometry. We showed that both MTO and microgel-loaded MTO penetrate into cells and both induce apoptosis and later secondary necrosis in a time- and dose dependent manner. In contrast, microgels without MTO are not cytotoxic in the corresponding concentrations. Our results show that MTO-loaded microgels are promising structures for application in magnetic drug targeting. - Highlights: • Densely packed spherical clusters of magnetic nanoparticles were obtained. • High magnetization microgels with superparamagnetic behavior are reported. • The facile and reproducible synthesis procedure applied is easy to be up-scaled. • The toxicity tests show that magnetic microgels are not cytotoxic. • We show that mitoxantrone loaded microgels induce death of Jurkat cells.

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

subtype identification in this novel, general model framework, and provide a detailed example using simulated data. We then describe application to a data set from an experimental study of antigen-specific T-cell subtyping using combinatorially encoded assays in human blood samples. Summary comments...... 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...

The use of bead beating to prepare suspensions of nuclei for flow cytometry from fresh leaves, herbarium leaves, petals and pollen.

Science.gov (United States)

Roberts, Andy V

2007-12-01

"Bead beating" is commonly used to release DNA from cells for genomic studies but it was used here to prepare suspensions of plant nuclei for measurement of DNA amounts by flow cytometry. Plant material was placed in 2-ml screw-capped tubes containing beads of zirconia/silica (2.5 mm diameter) or glass (2.5 or 1.0 mm diameter) and 1 ml of lysis buffer. The tubes were mechanically shaken with an FP120 FastPrep Cell Disrupter to release intact nuclei from plant tissue by the impact of the beads. The nuclei were then stained with propidium iodide (PI) and analyzed by flow cytometry. The method was tested using fresh leaves, fresh petals and herbarium leaves of Rosa canina, leaves and pollen of R. rugosa, and fresh leaves of Petroselinum crispum, Nicotiana tabacum, and Allium cepa. Batches of 12 samples of fresh leaves were prepared, simultaneously, in 45 s by bead beating in the Cell Disrupter. In flow cytometry histograms, nuclei of fresh leaves gave G(1)/G(0) peaks with CVs of less than 3.0% and nuclei from fresh petals and herbarium leaves of R. canina, and pollen of the generative nuclei of R. rugosa gave peaks with coefficients of variation (CVs) of less than 4.0%. DNA amounts estimated from 24-month-old herbarium leaves, using P. crispum as an internal standard, were less than those of fresh leaves by a small but significant amount. Suspensions of nuclei can be prepared rapidly and conveniently from a diversity of tissues by bead beating. Exposure of laboratory workers to harmful substances in the lysis buffer is minimized. (c) 2007 International Society for Analytical Cytology

Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

International Nuclear Information System (INIS)

Zhou Hongsheng; Zhang Donghua; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

2006-01-01

CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs

Organizing the Cellular and Molecular Heterogeneity in High-Grade Serous Ovarian Cancer by Mass Cytometry

Science.gov (United States)

2014-10-01

Bendall SC, Sung P, Nolan GP, Arvin AM. Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus. Cell Rep...Perspectives on Flow Cytometry 2013, September 20, 2013, Mass Cytometry and Cell Cycle, Mexico City, Mexico (by Web Conference) Nolan: Nuclear

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

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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

Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based viability detection method.

Science.gov (United States)

Chan, Leo Li-Ying; Kuksin, Dmitry; Laverty, Daniel J; Saldi, Stephanie; Qiu, Jean

2015-05-01

The ability to accurately determine cell viability is essential to performing a well-controlled biological experiment. Typical experiments range from standard cell culturing to advanced cell-based assays that may require cell viability measurement for downstream experiments. The traditional cell viability measurement method has been the trypan blue (TB) exclusion assay. However, since the introduction of fluorescence-based dyes for cell viability measurement using flow or image-based cytometry systems, there have been numerous publications comparing the two detection methods. Although previous studies have shown discrepancies between TB exclusion and fluorescence-based viability measurements, image-based morphological analysis was not performed in order to examine the viability discrepancies. In this work, we compared TB exclusion and fluorescence-based viability detection methods using image cytometry to observe morphological changes due to the effect of TB on dead cells. Imaging results showed that as the viability of a naturally-dying Jurkat cell sample decreased below 70 %, many TB-stained cells began to exhibit non-uniform morphological characteristics. Dead cells with these characteristics may be difficult to count under light microscopy, thus generating an artificially higher viability measurement compared to fluorescence-based method. These morphological observations can potentially explain the differences in viability measurement between the two methods.

New Method to Disaggregate and Analyze Single Isolated Helminthes Cells Using Flow Cytometry: Proof of Concept

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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.

Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder

Science.gov (United States)

Hattuniemi, Maarit; Korhonen, Johanna; Jaakkola, Mari; Räty, Jarkko; Virtanen, Vesa

2010-11-01

Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.

Flow cytometry of duodenal intraepithelial lymphocytes improves diagnosis of celiac disease in difficult cases.

Science.gov (United States)

Valle, Julio; Morgado, José Mario T; Ruiz-Martín, Juan; Guardiola, Antonio; Lopes-Nogueras, Miriam; García-Vela, Almudena; Martín-Sacristán, Beatriz; Sánchez-Muñoz, Laura

2017-10-01

Diagnosis of celiac disease is difficult when the combined results of serology and histology are inconclusive. Studies using flow cytometry of intraepithelial lymphocytes (IELs) have found that celiac patients have increased numbers of γδ IELs, along with a decrease in CD3-CD103 + IELs. The objective of this article is to assess the role of flow cytometric analysis of IELs in the diagnosis of celiac disease in difficult cases. A total of 312 patients with suspicion of celiac disease were included in the study. Duodenal biopsy samples were used for histological assessment and for flow cytometric analysis of IELs. In 46 out of 312 cases (14.7%) the combination of serology and histology did not allow the confirmation or exclusion of celiac disease. HLA typing had been performed in 42 of these difficult cases. Taking into account HLA typing and the response to a gluten-free diet, celiac disease was excluded in 30 of these cases and confirmed in the remaining 12. Flow cytometric analysis of IELs allowed a correct diagnosis in 39 out of 42 difficult cases (92.8%) and had a sensitivity of 91.7% (95% CI: 61.5% to 99.8%) and a specificity of 93.3% (95% CI: 77.9% to 99.2%) for the diagnosis of celiac disease in this setting. Flow cytometric analysis of IELs is useful for the diagnosis of celiac disease in difficult cases.

Flow cytometry apparatus

Science.gov (United States)

Pinkel, D.

1987-11-30

An obstruction across the flow chamber creates a one-dimensional convergence of a sheath fluid. A passageway in the obstruction directs flat cells near to the area of one-dimensional convergence in the sheath fluid to provide proper orientation of flat cells at fast rates. 6 figs.

Application of image cytometry to characterize heterologous lipid flippases in yeast

DEFF Research Database (Denmark)

Jensen, Maria Stumph; Costa, Sara; Theorin, Lisa

2016-01-01

Lipid flippases are integral membrane proteins that play a central role in moving lipids across cellular membranes. Some of these transporters are ATPases that couple lipid translocation to ATP hydrolysis, whereas others function without any discernible metabolic energy input. A growing number...... is typically monitored by flow cytometry, a costly and maintenance-intensive method. Here, we have optimized a protocol to use an automated image-based cell counter to accurately measure lipid uptake by heterologous lipid flippases expressed in yeast. The method was validated by comparison with the classical...... for characterization of lipid flippase activity, and should be readily adaptable to analyze a variety of other transport systems in yeast, parasites, and mammalian cells. © 2016 International Society for Advancement of Cytometry....

Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

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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.

Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death.

Science.gov (United States)

Viola, K S; Rodrigues, E M; Tanomaru-Filho, M; Carlos, I Z; Ramos, S G; Guerreiro-Tanomaru, J M; Faria, G

2017-01-30

To evaluate the cytotoxicity and the mechanism of cell aggression of peracetic acid (PA) in comparison with sodium hypochlorite (NaOCl). L929 fibroblasts were exposed to 1% PA and 2.5% NaOCl, at several dilutions for 10 min. The following parameters were evaluated: cell metabolism by methylthiazol tetrazolium assay, external morphology by scanning electron microscopy, ultrastructure by transmission electron microscopy, the cytoskeleton by means of actin and α-tubulin labelling, and the type of cell death by flow cytometry (apoptosis/necrosis). The data were analysed by two-way anova and the Bonferroni post-test (α = 0.05). The PA group had lower cell viability and a higher percentage of necrotic cells than the NaOCl group (P < 0.05). Both solutions diminished cell metabolism, led to destructuring of the cytoskeleton, created changes in the external morphology, resulted in the accumulation of proteins in the rough endoplasmic reticulum and induced cell death predominantly by necrosis. However, these changes were observed in lower doses of PA when compared with NaOCl. Although they had the same mechanism of cytotoxicity, 1% PA had greater cytotoxic potential than 2.5% NaOCl. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Cytobank: providing an analytics platform for community cytometry data analysis and collaboration.

Science.gov (United States)

Chen, Tiffany J; Kotecha, Nikesh

2014-01-01

Cytometry is used extensively in clinical and laboratory settings to diagnose and track cell subsets in blood and tissue. High-throughput, single-cell approaches leveraging cytometry are developed and applied in the computational and systems biology communities by researchers, who seek to improve the diagnosis of human diseases, map the structures of cell signaling networks, and identify new cell types. Data analysis and management present a bottleneck in the flow of knowledge from bench to clinic. Multi-parameter flow and mass cytometry enable identification of signaling profiles of patient cell samples. Currently, this process is manual, requiring hours of work to summarize multi-dimensional data and translate these data for input into other analysis programs. In addition, the increase in the number and size of collaborative cytometry studies as well as the computational complexity of analytical tools require the ability to assemble sufficient and appropriately configured computing capacity on demand. There is a critical need for platforms that can be used by both clinical and basic researchers who routinely rely on cytometry. Recent advances provide a unique opportunity to facilitate collaboration and analysis and management of cytometry data. Specifically, advances in cloud computing and virtualization are enabling efficient use of large computing resources for analysis and backup. An example is Cytobank, a platform that allows researchers to annotate, analyze, and share results along with the underlying single-cell data.

Rapid Identification of Staphylococcus aureus and Methicillin Resistance by Flow Cytometry Using a Peptide Nucleic Acid Probe ▿

Science.gov (United States)

Shrestha, Nabin K.; Scalera, Nikole M.; Wilson, Deborah A.; Brehm-Stecher, Byron; Procop, Gary W.

2011-01-01

A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively. PMID:21795508

Differences in leukocyte differentiation molecule abundances on domestic sheep (Ovis aries) and bighorn sheep (Ovis canadensis) neutrophils identified by flow cytometry.

Science.gov (United States)

Highland, Margaret A; Schneider, David A; White, Stephen N; Madsen-Bouterse, Sally A; Knowles, Donald P; Davis, William C

2016-06-01

Although both domestic sheep (DS) and bighorn sheep (BHS) are affected by similar respiratory bacterial pathogens, experimental and field data indicate BHS are more susceptible to pneumonia. Cross-reactive monoclonal antibodies (mAbs) for use in flow cytometry (FC) are valuable reagents for interspecies comparative immune system analyses. This study describes cross-reactive mAbs that recognize leukocyte differentiation molecules (LDMs) and major histocompatibility complex antigens on DS and BHS leukocytes. Characterization of multichannel eosinophil autofluorescence in this study permitted cell-type specific gating of granulocytes for evaluating LDMs, specifically on neutrophils, by single-label FC. Evaluation of relative abundances of LDMs by flow cytometry revealed greater CD11a, CD11b, CD18 (β2 integrins) and CD 172a (SIRPα) on DS neutrophils and greater CD14 (lipopolysaccharide receptor) on BHS neutrophils. Greater CD25 (IL-2) was identified on BHS lymphocytes following Concavalin A stimulation. While DS and BHS have similar total peripheral blood leukocyte counts, BHS have proportionately more neutrophils. Published by Elsevier Ltd.

The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells

International Nuclear Information System (INIS)

Li, Jun; Qin, Zhenghong; Liang, Zhongqin

2009-01-01

Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells

SCENERY: a web application for (causal) network reconstruction from cytometry data

KAUST Repository

Papoutsoglou, Georgios

2017-05-08

Flow and mass cytometry technologies can probe proteins as biological markers in thousands of individual cells simultaneously, providing unprecedented opportunities for reconstructing networks of protein interactions through machine learning algorithms. The network reconstruction (NR) problem has been well-studied by the machine learning community. However, the potentials of available methods remain largely unknown to the cytometry community, mainly due to their intrinsic complexity and the lack of comprehensive, powerful and easy-to-use NR software implementations specific for cytometry data. To bridge this gap, we present Single CEll NEtwork Reconstruction sYstem (SCENERY), a web server featuring several standard and advanced cytometry data analysis methods coupled with NR algorithms in a user-friendly, on-line environment. In SCENERY, users may upload their data and set their own study design. The server offers several data analysis options categorized into three classes of methods: data (pre)processing, statistical analysis and NR. The server also provides interactive visualization and download of results as ready-to-publish images or multimedia reports. Its core is modular and based on the widely-used and robust R platform allowing power users to extend its functionalities by submitting their own NR methods. SCENERY is available at scenery.csd.uoc.gr or http://mensxmachina.org/en/software/.

Novel Strategy for Phenotypic Characterization of Human B Lymphocytes from Precursors to Effector Cells by Flow Cytometry.

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Giovanna Clavarino

Full Text Available 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.

Evaluation of chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effects of Aloysia citrodora extract on colon cancer cell line

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Amir Mirzaie

2016-06-01

Full Text Available Background: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer (HT29 cells by using real-time polymerase chain reaction and flow-cytometry methods. Methods: This experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH, disk diffusion and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT methods, respectively. The half maximal inhibitory concentration (IC50 value was calculated. We extracted total RNA molecules by using RNX solution, after which cDNA was synthesized. Finally, the pro-apoptotic (Bax and anti-apoptotic (Bcl2 gene expression was performed by real-time polymerase chain reaction and apoptotic effects were analyzed using Flow-cytometry method. Results: GC-MS analysis of Aloysia citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57% and Caryophyllene oxide (15.15% The antioxidant activity of the extract was IC50= 0.6±0.03 mg/ml. The maximum and minimum antibacterial effects of extract were belonged to gram-negative and gram-positive bacteria, respectively. Cytotoxic results revealed that the A.citrodora extract have IC50= 20.1±0.78 mg/ml against colon cancer (HT29 cell line and real-time polymerase chain reaction results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470±0.72 (P< 0.05, 0.43±0.35 (P< 0.05. In addition, the flow-cytometry results indicated the 38

Cytotoxicity and Apoptotic Effects of Polyphenols from Sugar Beet Molasses on Colon Carcinoma Cells in Vitro

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Mingshun Chen

2016-06-01

Full Text Available Three polyphenols were isolated and purified from sugar beet molasses by ultrasonic-aid extraction and various chromatographic techniques, and their structures were elucidated by spectral analysis. Cytotoxicity and the molecular mechanism were measured by methyl thiazolyl tetrazolium (MTT assay, flow cytometry, caspase-3 activity assay and Western blot assay. The results showed that gallic acid, cyanidin-3-O-glucoside chloride and epicatechin have cytotoxicity to the human colon, hepatocellular and breast cancer cells. Cyanidin-3-O-glucoside chloride showed its cytotoxicity against various tumor cell lines, particularly against colon cancer Caco-2 cells with half maximal inhibitory concentration (IC50 value of 23.21 ± 0.14 μg/mL in vitro. Cyanidin-3-O-glucoside chloride may be a potential candidate for the treatment of colon cancer. In the mechanism study, cyanidin-3-O-glucoside chloride increased the ratio of cell cycle at G0/G1 phase and reduced cyclin D1 expression on Caco-2 cells. Cyanidin-3-O-glucoside chloride decreased mutant p21 expression, and increased the ratio of Bax/Bcl-2 and the activation of caspase-3 to induce apoptosis.

Involvement of enniatins-induced cytotoxicity in human HepG2 cells.

Science.gov (United States)

Juan-García, Ana; Manyes, Lara; Ruiz, María-José; Font, Guillermina

2013-04-12

Enniatins (ENNs) are mycotoxins found in Fusarium fungi and they appear in nature as mixtures of cyclic depsipeptides. The ability to form ionophores in the cell membrane is related to their cytotoxicity. Changes in ion distribution between inner and outer phases of the mitochondria affect to their metabolism, proton gradient, and chemiosmotic coupling, so a mitochondrial toxicity analysis of enniatins is highly recommended because they host the homeostasis required for cellular survival. Two ENNs, ENN A and ENN B on hepatocarcinoma cells (HepG2) at 1.5 and 3 μM and three exposure times (24, 48 and 72 h) were studied. Flow cytometry was used to examine their effects on cell proliferation, to characterize at which phase of the cell cycle progression the cells were blocked and to study the role of the mitochondrial in ENNs-induced apoptosis. In conclusion, apoptosis induction on HepG2 cells allowed to compare cytotoxic effects caused by both ENNs, A and B. It is reported the possible mechanism observed in MMP changes, cell cycle analysis and apoptosis/necrosis, identifying ENN B more toxic than ENN A. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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.

Flow cytometry and integrated imaging

Directory of Open Access Journals (Sweden)

V. Kachel

2000-06-01

Full Text Available It is a serious problem to relate the results of a flow cytometric analysis of a marine sample to different species. Images of particles selectively triggered by the flow cytometric analysis and picked out from the flowing stream give a valuable additional information on the analyzed organisms. The technical principles and problems of triggered imaging in flow are discussed, as well as the positioning of the particles in the plane of focus, freezing the motion of the quickly moving objects and what kinds of light sources are suitable for pulsed illumination. The images have to be stored either by film or electronically. The features of camera targets and the memory requirements for storing the image data and the conditions for the triggering device are shown. A brief explanation of the features of three realized flow cytometric imaging (FCI systems is given: the Macro Flow Planktometer built within the EUROMAR MAROPT project, the Imaging Module of the European Plankton Analysis System, supported by the MAST II EurOPA project and the most recently developed FLUVO VI universal flow cytometer including HBO 100- and laser excitation for fluorescence and scatter, Coulter sizing as well as bright field and and phase contrast FCI.

Evaluating the effects of allelochemical ferulic acid on Microcystis aeruginosa by pulse-amplitude-modulated (PAM) fluorometry and flow cytometry.

Science.gov (United States)

Wang, Rui; Hua, Ming; Yu, Yang; Zhang, Min; Xian, Qi-Ming; Yin, Da-Qiang

2016-03-01

We investigated the effects of allelochemical ferulic acid (FA) on a series of physiological and biochemical processes of blue-green algae Microcystis aeruginosa, in order to find sensitive diagnostic variables for allelopathic effects. Algal cell density was significantly suppressed by FA (0.31-5.17 mM) only after 48 h exposure. Inhibitions of photosynthetic parameters (F(v)/F(m) and F(v)'/F(m)') occurred more rapidly than cell growth, and the stimulation of non-photochemical quenching was observed as a feed-back mechanisms induced by photosystem II blockage, determining by PAM fluorometry. Inhibitions on esterase activity, membrane potential and integrity, as well as disturbance on cell size, were all detected by flow cytometry with specific fluorescent markers, although exhibiting varied sensitivities. Membrane potential and esterase activity were identified as the most sensitive parameters (with relatively lower EC50 values), and responded more rapidly (significantly inhibited only after 8 h exposure) than photosynthetic parameters and cell growth, thus may be the primary responses of cyanobacteria to FA exposure. The use of PAM fluorometry and flow cytometry for rapid assessment of those sensitive variables may contribute to future mechanistic studies of allolepathic effects on phytoplankton. Copyright © 2015 Elsevier Ltd. All rights reserved.

A fluorescence-based rapid screening assay for cytotoxic compounds

International Nuclear Information System (INIS)

Montoya, Jessica; Varela-Ramirez, Armando; Estrada, Abril; Martinez, Luis E.; Garza, Kristine; Aguilera, Renato J.

2004-01-01

A simple fluorescence-based assay was developed for the rapid screening of potential cytotoxic compounds generated by combinatorial chemistry. The assay is based on detection of nuclear green fluorescent protein (GFP) staining of a human cervical cancer cell line (HeLa) carrying an integrated histone H2B-GFP fusion gene. Addition of a cytotoxic compound to the HeLa-GFP cells results in the eventual degradation of DNA and loss of the GFP nuclear fluorescence. Using this assay, we screened 11 distinct quinone derivatives and found that several of these compounds were cytotoxic. These compounds are structurally related to plumbagin an apoptosis-inducing naphthoquinone isolated from Black Walnut. In order to determine the mechanism by which cell death was induced, we performed additional experiments with the most cytotoxic quinones. These compounds were found to induce morphological changes (blebbing and nuclear condensation) consistent with induction of apoptosis. Additional tests revealed that the cytotoxic compounds induce both necrotic and apoptotic modes of death

Anaplasma phagocytophilum-Related Defects in CD8, NKT, and NK Lymphocyte Cytotoxicity

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Diana G. Scorpio

2018-04-01

Full Text Available Human granulocytic anaplasmosis, caused by the tick-transmitted Anaplasma phagocytophilum, is not controlled by innate immunity, and induces a proinflammatory disease state with innate immune cell activation. In A. phagocytophilum murine infection models, hepatic injury occurs with production of IFNγ thought to be derived from NK, NKT cells, and CD8 T lymphocytes. Specific A. phagocytophilum ligands that drive inflammation and disease are not known, but suggest a clinical and pathophysiologic basis strikingly like macrophage activation syndrome (MAS and hemophagocytic syndrome (HPS. We studied in vivo responses of NK, NKT, and CD8 T lymphocytes from infected animals for correlates of lymphocyte-mediated cytotoxicity and examined in vitro interactions with A. phagocytophilum-loaded antigen-presenting cells (APCs. Murine splenocytes were examined and found deficient in cytotoxicity as determined by CD107a expression in vitro for specific CTL effector subsets as determined by flow cytometry. Moreover, A. phagocytophilum-loaded APCs did not lead to IFNγ production among CTLs in vitro. These findings support the concept of impaired cytotoxicity with A. phagocytophilum presentation by APCs that express MHC class I and that interact with innate and adaptive immune cells with or after infection. The findings strengthen the concept of an enhanced proinflammatory phenotype, such as MAS and HPS disease states as the basis of disease and severity with A. phagocytophilum infection, and perhaps by other obligate intracellular bacteria.

Two new nuclear isolation buffers for plant DNA flow cytometry: A test with 37 species

Czech Academy of Sciences Publication Activity Database

Loureiro, J.; Rodriguez, E.; Doležel, Jaroslav; Santos, C.

2007-01-01

Roč. 100, č. 4 (2007), s. 875-888 ISSN 0305-7364 R&D Projects: GA ČR GA521/06/1723; GA ČR(CZ) GA521/05/0257; GA MŠk(CZ) LC06004 Grant - others:Mendelova zemědělská a lesnická univerzita v Brně / Agronomická fakulta(CZ) ME 844 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje ; V - iné verejné zdroje ; V - iné verejné zdroje ; V - iné verejné zdroje Keywords : flow cytometry * general purpose buffer * genome size Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.939, year: 2007

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

Science.gov (United States)

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

Rapamycin potentiates cytotoxicity by docetaxel possibly through downregulation of Survivin in lung cancer cells

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Li Hui

2011-03-01

Full Text Available Abstract Background To elucidate whether rapamycin, the inhibitor of mTOR (mammalian target of rapamycin, can potentiate the cytotoxic effect of docetaxel in lung cancer cells and to probe the mechanism underlying such enhancement. Methods Lung cancer cells were treated with docetaxel and rapamycin. The effect on the proliferation of lung cancer cells was evaluated using the MTT method, and cell apoptosis was measured by flow cytometry. Protein expression and level of phosphorylation were assayed using Western Blot method. Results Co-treatment of rapamycin and docetaxel was found to favorably enhance the cytotoxic effect of docetaxel in four lung cancer cell lines. This tumoricidal boost is associated with a reduction in the expression and phosphorylation levels of Survivin and ERK1/2, respectively. Conclusion The combined application of mTOR inhibitor and docetaxel led to a greater degree of cancer cell killing than that by either compound used alone. Therefore, this combination warrants further investigation in its suitability of serving as a novel therapeutic scheme for treating advanced and recurrent lung cancer patients.

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

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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

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

Science.gov (United States)

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor

Performance standard-based validation study for local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method.

Science.gov (United States)

Ahn, Ilyoung; Kim, Tae-Sung; Jung, Eun-Sun; Yi, Jung-Sun; Jang, Won-Hee; Jung, Kyoung-Mi; Park, Miyoung; Jung, Mi-Sook; Jeon, Eun-Young; Yeo, Kyeong-Uk; Jo, Ji-Hoon; Park, Jung-Eun; Kim, Chang-Yul; Park, Yeong-Chul; Seong, Won-Keun; Lee, Ai-Young; Chun, Young Jin; Jeong, Tae Cheon; Jeung, Eui Bae; Lim, Kyung-Min; Bae, SeungJin; Sohn, Soojung; Heo, Yong

2016-10-01

Local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) is a modified non-radioisotopic technique with the additional advantages of accommodating multiple endpoints with the introduction of FCM, and refinement and reduction of animal use by using a sophisticated prescreening scheme. Reliability and accuracy of the LLNA: BrdU-FCM was determined according to OECD Test Guideline (TG) No. 429 (Skin Sensitization: Local Lymph Node Assay) performance standards (PS), with the participation of four laboratories. Transferability was demonstrated through successfully producing stimulation index (SI) values for 25% hexyl cinnamic aldehyde (HCA) consistently greater than 3, a predetermined threshold, by all participating laboratories. Within- and between-laboratory reproducibility was shown using HCA and 2,4-dinitrochlorobenzene, in which EC2.7 values (the estimated concentrations eliciting an SI of 2.7, the threshold for LLNA: BrdU-FCM) fell consistently within the acceptance ranges, 0.025-0.1% and 5-20%, respectively. Predictive capacity was tested using the final protocol version 1.3 for the 18 reference chemicals listed in OECD TG 429, of which results showed 84.6% sensitivity, 100% specificity, and 88.9% accuracy compared with the original LLNA. The data presented are considered to meet the performance criteria for the PS, and its predictive capacity was also sufficiently validated. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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

Absence of DNA double-strand breaks in human peripheral blood mononuclear cells after 3 Tesla magnetic resonance imaging assessed by γH2AX flow cytometry

Energy Technology Data Exchange (ETDEWEB)

Fasshauer, Martin; Staab, Wieland; Sohns, Jan M.; Ritter, Christian; Lotz, Joachim [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Kruewel, Thomas; Stahnke, Vera C. [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); Zapf, Antonia [University Medical Center Goettingen, Department of Medical Statistics, Goettingen (Germany); Rave-Fraenk, Margret [University Medical Center Goettingen, Department of Radiotherapy and Radiooncology, Goettingen (Germany); Steinmetz, Michael [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Pediatric Cardiology and Intensive Care Medicine, University Medical Center Goettingen (Germany); Unterberg-Buchwald, Christina [Goettingen Heart Center, Department of Diagnostic and Interventional Radiology, University Medical Center Goettingen (Germany); German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany); Schuster, Andreas [German Centre for Cardiovascular Research (DZHK), Goettingen (Germany); Goettingen Heart Center, Department of Cardiology and Pneumology, University Medical Center Goettingen (Germany)

2018-03-15

Magnetic resonance imaging (MRI) is regarded as a non-harming and non-invasive imaging modality with high tissue contrast and almost no side effects. Compared to other cross-sectional imaging modalities, MRI does not use ionising radiation. Recently, however, strong magnetic fields as applied in clinical MRI scanners have been suspected to induce DNA double-strand breaks in human lymphocytes. In this study we investigated the impact of 3-T cardiac MRI examinations on the induction of DNA double-strand breaks in peripheral mononuclear cells by γH2AX staining and flow cytometry analysis. The study cohort consisted of 73 healthy non-smoking volunteers with 36 volunteers undergoing CMRI and 37 controls without intervention. Differences between the two cohorts were analysed by a mixed linear model with repeated measures. Both cohorts showed a significant increase in the γH2AX signal from baseline to post-procedure of 6.7 % (SD 7.18 %) and 7.8 % (SD 6.61 %), respectively. However, the difference between the two groups was not significant. Based on our study, γH2AX flow cytometry shows no evidence that 3-T MRI examinations as used in cardiac scans impair DNA integrity in peripheral mononuclear cells. (orig.)

Gross genomic damage measured by DNA image cytometry independently predicts gastric cancer patient survival

NARCIS (Netherlands)

Belien, J.A.M.; Buffart, T.E.; Gill, A.; Broeckaert, M.A.M.; Quirke, P.; Meijer, G.A.; Grabsch, H.

2009-01-01

BACKGROUND: DNA aneuploidy reflects gross genomic changes. It can be measured by flow cytometry (FCM-DNA) or image cytometry (ICM-DNA). In gastric cancer, the prevalence of DNA aneuploidy has been reported to range from 27 to 100%, with conflicting associations with clinicopathological variables.

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

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Rok Gaber

2013-11-01

Full Text Available To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

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Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

2013-01-01

To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

Evaluation and SAR analysis of the cytotoxicity of tanshinones in colon cancer cells.

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Wang, Lin; Liu, An; Zhang, Fei-Long; Yeung, John H K; Li, Xu-Qin; Cho, Chi-Hin

2014-03-01

This study was designed to evaluate the anti-cancer actions of tanshinone I and tanshinone IIA, and six derivatives of tanshinone IIA on normal and cancerous colon cells. Structure activity relationship (SAR) analysis was conducted to delineate the significance of the structural modifications of tanshinones for improved anti-cancer action. Tanshinone derivatives were designed and synthesized according to the literature. The cytotoxicity of different compounds on colon cancer cells was determined by the MTT assay. Apoptotic activity of the tanshinones was measured by flow cytometry (FCM). Tanshinone I and tanshinone IIA both exhibited significant cytotoxicity on colon cancer cells. They are more effective in p53(+/+) colon cancer cell line. It was also noted that the anti-cancer activity of tanshinone I was more potent and selective. Two of the derivatives of tanshinone IIA (N1 and N2) also exhibited cytotoxicity on colon cancer cells. The anti-colon cancer activity of tanshinone I was more potent and selective than tanshinone IIA, and is p53 dependent. The derivatives obtained by structural modifications of tanshinone IIA exhibited lower cytotoxicity on both normal and colon cancer cells. From steric and electronic characteristics point of view, it was concluded that structural modifications of ring A and furan or dihydrofuran ring D on the basic structure of tanshinones influences the activity. An increase of the delocalization of the A and B rings could enhance the cytotoxicity of such compounds, while a non-planar and small sized D ring region would provide improved anti-cancer activity. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma

International Nuclear Information System (INIS)

Silvennoinen, R; Lundan, T; Kairisto, V; Pelliniemi, T-T; Putkonen, M; Anttila, P; Huotari, V; Mäntymaa, P; Siitonen, S; Uotila, L; Penttilä, T-L; Juvonen, V; Selander, T; Remes, K

2014-01-01

Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6−10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities

Cytotoxic and toxicological effects of phthalimide derivatives on tumor and normal murine cells

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PAULO MICHEL PINHEIRO FERREIRA

2015-03-01

Full Text Available Eleven phthalimide derivatives were evaluated with regards to their antiproliferative activity on tumor and normal cells and possible toxic effects. Cytotoxic analyses were performed against murine tumors (Sarcoma 180 and B-16/F-10 cells and peripheral blood mononuclear cells (PBMC using MTT and Alamar Blue assays. Following, the investigation of cytotoxicity was executed by flow cytometry analysis and antitumoral and toxicological potential by in vivo techniques. The molecules 3b, 3c, 4 and 5 revealed in vitro cytotoxicity against Sarcoma 180, B-16/F-10 and PBMC. Since compound 4 was the most effective derivative, it was chosen to detail the mechanism of action after 24, 48 and 72 h exposure (22.5 and 45 µM. Sarcoma 180 cells treated with compound 4 showed membrane disruption, DNA fragmentation and mitochondrial depolarization in a time- and dose-dependent way. Compounds 3c, 4 and 5 (50 mg/kg/day did not inhibit in vivotumor growth. Compound 4-treated animals exhibited an increase in total leukocytes, lymphocytes and spleen relative weight, a decreasing in neutrophils and hyperplasia of spleen white pulp. Treated animals presented reversible histological changes. Molecule 4 had in vitro antiproliferative action possibly triggered by apoptosis, reversible toxic effects on kidneys, spleen and livers and exhibited immunostimulant properties that can be explored to attack neoplasic cells.

Measuring cell cycle progression kinetics with metabolic labeling and flow cytometry.

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Fleisig, Helen; Wong, Judy

2012-05-22

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments. Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key

Effect of fluoride-treated enamel on indirect cytotoxicity of a 16% carbamide peroxide bleaching gel to pulp cells.

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Soares, Diana Gabriela; Ribeiro, Ana Paula Dias; Lima, Adriano Fonseca; Sacono, Nancy Tomoko; Hebling, Josimeri; de Souza Costa, Carlos Alberto

2013-01-01

The aim of this study was to evaluate the possibility of fluoride solutions applied to enamel to protect pulp cells against the trans-enamel and transdentinal cytotoxicity of a 16% carbamide peroxide (CP) bleaching gel. The CP gel was applied to enamel/dentin discs adapted to aicial pulp chambers (8 h/day) during 1, 7 or 14 days, followed by fluoride (0.05% or 0.2%) application for 1 min. The extracts (culture medium in contact with dentin) were applied to MDPC-23 cells for 1 h, and cell metabolism (MTT assay), alkaline phosphatase (ALP) activity and cell membrane damage (flow cytometry) were analyzed. Knoop microhardness of enamel was also evaluated. Data were analyzed statistically by ANOVA and Kruskal-Wallis tests (α=0.05). For the MTT assay and ALP activity, significant reductions between the control and the bleached groups were observed (p0.05), regardless of fluoride application or treatment days. Flow cytometry analysis demonstrated 30% of cell membrane damage in all bleached groups. After 14 days of treatment, the fluoride-treated enamel presented significantly higher microhardness values than the bleached-only group (pfluoride solutions, the treated enamel surface did not prevent the toxic effects caused by the 16% CP gel to odontoblast-like cells.

Validation of Microcapillary Flow Cytometry for Community-Based CD4+ T Lymphocyte Enumeration in Remote Burkina Faso

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Renault, Cybèle A; Traore, Arouna; Machekano, Rhoderick N; Israelski, Dennis M

2010-01-01

Background: CD4+ T lymphocyte enumeration plays a critical role in the initiation and monitoring of HIV-infected patients on antiretroviral therapy. There is an urgent need for low-cost CD4+ enumeration technologies, particularly for use in dry, dusty climates characteristic of many small cities in Sub-Saharan Africa. Design: Cross-sectional study. Methods: Blood samples from 98 HIV-infected patients followed in a community HIV clinic in Ouahigouya, Burkina Faso were obtained for routine CD4+ T lymphocyte count monitoring. The blood samples were divided into two aliquots, on which parallel CD4+ measurements were performed using microcapillary (Guava EasyCD4) and dedicated (Becton Dickinson FACSCount) CD4+ enumeration systems. Spearman rank correlation coefficient was calculated, and the sensitivity, specificity and positive predictive value (PPV) for EasyCD4 <200 cells/µL were determined compared to the reference standard FACSCount CD4 <200 cells/µL. Results: Mean CD4 counts for the EasyCD4 and FACSCount were 313.75 cells/µL and 303.47 cells/µL, respectively. The Spearman rank correlation coefficient was 0.92 (p<0.001). Median values using EasyCD4 were higher than those with the FACSCount (p=0.004). For a CD4<350 cells/uL, sensitivity of the EasyCD4 was 93.9% (95%CI 85.2-98.3%), specificity was 90.6% (95% CI 75.0-98.0%), and PPV was 95.4% (95%CI 87.1-99.0%). Conclusion: Use of the EasyCD4 system was feasible and highly accurate in the harsh conditions of this remote city in Sub-Saharan Africa, demonstrating acceptable sensitivity and specificity compared to a standard operating system. Microcapillary flow cytometry offers a cost-effective alternative for community-based, point-of-care CD4+ testing and could play a substantial role in scaling up HIV care in remote, resource-limited settings. PMID:21253463

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

Science.gov (United States)

Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Measurement of Soluble Biomarkers by Flow Cytometry

OpenAIRE

Antal-Szalm?s, P?ter; Nagy, B?la; Debreceni, Ildik? Beke; Kappelmayer, J?nos

2013-01-01

Microparticle based flow cytometric assays for determination of the level of soluble biomarkers are widely used in several research applications and in some diagnostic setups. The major advantages of these multiplex systems are that they can measure a large number of analytes (up to 500) at the same time reducing assay time, costs and sample volume. Most of these assays are based on antigen-antibody interactions and work as traditional immunoassays, but nucleic acid alterations ? by using spe...

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.

Enumeration of antigen-specific CD8+ T lymphocytes by single-platform, HLA tetramer-based flow cytometry: a European multicenter evaluation.

Science.gov (United States)

Heijnen, Ingmar A F M; Barnett, David; Arroz, Maria J; Barry, Simon M; Bonneville, Marc; Brando, Bruno; D'hautcourt, Jean-Luc; Kern, Florian; Tötterman, Thomas H; Marijt, Erik W A; Bossy, David; Preijers, Frank W M B; Rothe, Gregor; Gratama, Jan W

2004-11-01

HLA class I peptide tetramers represent powerful diagnostic tools for detection and monitoring of antigen-specific CD8(+) T cells. The impetus for the current multicenter study is the critical need to standardize tetramer flow cytometry if it is to be implemented as a routine diagnostic assay. Hence, the European Working Group on Clinical Cell Analysis set out to develop and evaluate a single-platform tetramer-based method that used cytomegalovirus (CMV) as the antigenic model. Absolute numbers of CMV-specific CD8(+) T cells were obtained by combining the percentage of tetramer-binding cells with the absolute CD8(+) T-cell count. Six send-outs of stabilized blood from healthy individuals or CMV-carrying donors with CMV-specific CD8(+) T-cell counts of 3 to 10 cells/microl were distributed to 7 to 16 clinical sites. These sites were requested to enumerate CD8(+) T cells and, in the case of CMV-positive donors, CMV-specific subsets on three separate occasions using the standard method. Between-site coefficients of variation of less than 10% (absolute CD8(+) T-cell counts) and approximately 30% (percentage and absolute numbers of CMV-specific CD8(+) T cells) were achieved. Within-site coefficients of variation were approximately 5% (absolute CD8(+) T-cell counts), approximately 9% (percentage CMV-specific CD8(+) T cells), and approximately 17% (absolute CMV-specific CD8(+) T-cell counts). The degree of variation tended to correlate inversely with the proportion of CMV-specific CD8(+) T-cell subsets. The single-platform MHC tetramer-based method for antigen-specific CD8(+) T-cell counting has been evaluated by a European group of laboratories and can be considered a reproducible assay for routine enumeration of antigen-specific CD8(+) T cells. (c) 2004 Wiley-Liss, Inc.

Isolation and Flow Cytometry Analysis of Innate Lymphoid Cells from the Intestinal Lamina Propria.

Science.gov (United States)

Gronke, Konrad; Kofoed-Nielsen, Michael; Diefenbach, Andreas

2017-01-01

The intestinal mucosa constitutes the biggest surface area of the body. It is constantly challenged by bacteria, commensal and pathogenic, protozoa, and food-derived irritants. In order to maintain homeostasis, a complex network of signaling circuits has evolved that includes contributions of immune cells. In recent years a subset of lymphocytes, which belong to the innate immune system, has caught particular attention. These so-called innate lymphoid cells (ILC) reside within the lamina propria of the small and large intestines and rapidly respond to environmental challenges. They provide immunity to various types of infections but may also contribute to organ homeostasis as they produce factors acting on epithelial cells thereby enhancing barrier integrity. Here, we describe how these cells can be isolated from their environment and provide an in-depth protocol how to visualize the various ILC subsets by flow cytometry.

A study of light scattering of mononuclear blood cells with scanning flow cytometry

International Nuclear Information System (INIS)

Zharinov, Alexey; Tarasov, Peter; Shvalov, Alexander; Semyanov, Konstantin; Bockstaele, Dirk R. van; Maltsev, Valeri

2006-01-01

This study describes the measurement of light scattering of human mononuclear blood cells, the development of an appropriate optical model for those cells, and solution of the inverse light-scattering problem. The angular dependency of light-scattering intensity of mononuclear blood cells was experimentally measured by means of scanning flow cytometry. A sphere consisting of several concentric homogeneous layers with different refractive indices was tested as an optical model for mononuclear blood cells. A five-layer model has given the best agreement between experimental and theoretical light-scattering profiles. The inverse light-scattering problem was solved for a five-layer model with an optimization procedure that allows one to retrieve cell parameters: cell size relates to the outer diameter of the fifth layer; size of the nucleus relates to the outer diameter of the third layer. Mean values of cell size, nuclear size, refractive indices of nucleus and cellular cytoplasm were determined for blood monocytes and lymphocytes

Dendritic cells induce specific cytotoxic T lymphocytes against prostate cancer TRAMP-C2 cells loaded with freeze- thaw antigen and PEP-3 peptide.

Science.gov (United States)

Liu, Xiao-Qi; Jiang, Rong; Li, Si-Qi; Wang, Jing; Yi, Fa-Ping

2015-01-01

Prostate cancer is the most common cancer in men. In this study, we investigated immune responses of cytotoxic T lymphocytes (CTLs) against TRAMP-C2 prostate cancer cells after activation by dendritic cells (DCs) loaded with TRAMP-C2 freeze-thaw antigen and/or PEP-3 peptide in vitro. Bone marrow-derived DC from the bone marrow of the C57BL/6 were induced to mature by using the cytokine of rhGM-CSF and rhIL-4, and loaded with either the freeze-thaw antigen or PEP-3 peptide or both of them. Maturation of DCs was detected by flow cytometry. The killing efficiency of the CTLs on TRAMP-C2 cells were detected by flow cytometry, CCK8, colony formation, transwell migration, and wound-healing assay. The levels of the IFN-γ, TNF-β and IL-12 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with the unloaded DCs, the loaded DCs had significantly increased expression of several phenotypes related to DC maturation. CTLs activated by DCs loaded with freeze-thaw antigen and PEP-3 peptide had more evident cytotoxicity against TRAMP-C2 cells in vitro. The secretion levels of IFN-γ, TNF-β and IL-12, secreted by DCs loaded with antigen and PEP-3 and interaction with T cells, were higher than in the other groups. Our results suggest that the CTLs activated by DCs loaded with TRAMP-C2 freeze-thaw antigen and PEP-3 peptide exert a remarkable killing efficiency against TRAMP-C2 cells in vitro.

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.

Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

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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.

Toxic Effects of Ethyl Cinnamate on the Photosynthesis and Physiological Characteristics of Chlorella vulgaris Based on Chlorophyll Fluorescence and Flow Cytometry Analysis

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Jiao, Yang; Ouyang, Hui-Ling; Jiang, Yu-Jiao; Kong, Xiang-Zhen; He, Wei; Liu, Wen-Xiu; Yang, Bin; Xu, Fu-Liu

2015-01-01

The toxic effects of ethyl cinnamate on the photosynthetic and physiological characteristics of Chlorella vulgaris were studied based on chlorophyll fluorescence and flow cytometry analysis. Parameters, including biomass, F v/F m (maximal photochemical efficiency of PSII), ФPSII (actual photochemical efficiency of PSII in the light), FDA, and PI staining fluorescence, were measured. The results showed the following: (1) The inhibition on biomass increased as the exposure concentration increased. 1 mg/L ethyl cinnamate was sufficient to reduce the total biomass of C. vulgaris. The 48-h and 72-h EC50 values were 2.07 mg/L (1.94–2.20) and 1.89 mg/L (1.82–1.97). (2) After 24 h of exposure to 2–4 mg/L ethyl cinnamate, the photosynthesis of C. vulgaris almost ceased, manifesting in ФPSII being close to zero. After 72 h of exposure to 4 mg/L ethyl cinnamate, the F v/F m of C. vulgaris dropped to zero. (3) Ethyl cinnamate also affected the cellular physiology of C. vulgaris, but these effects resulted in the inhibition of cell yield rather than cell death. Exposure to ethyl cinnamate resulted in decreased esterase activities in C. vulgaris, increased average cell size, and altered intensities of chlorophyll a fluorescence. Overall, esterase activity was the most sensitive variable. PMID:26101784

Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

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Yuting Guo

2017-07-01

Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

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Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu; Scheuermann, Richard H

2016-01-01

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell

Cytotoxicity and apoptosis induction by e-cigarette fluids in human gingival fibroblasts.

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Sancilio, Silvia; Gallorini, Marialucia; Cataldi, Amelia; di Giacomo, Viviana

2016-04-01

Electronic cigarettes (e-cigarettes) are generally acknowledged as a safer alternative to the use of combusted tobacco products. Nevertheless, there are increasing conflicting claims concerning the effect of these novel industrial products on the health of e-cigarettes users. The aim of this work was to investigate the effects of the liquids of e-cigarettes on human gingival fibroblasts (HGFs) and to compare the effects of nicotine-containing fluid to the fluid itself. HGFs were treated with different concentrations (0-5 mg/mL) of fluids of e-cigarettes for different times (0-72 h) and cytotoxicity was analyzed by MTT assay. Fluids were administered also after being vaped (e.g., warmed into the cartomizer). Apoptosis occurrence and Bax expression were evaluated by flow cytometry; ROS production was analyzed by fluorescence optical microscopy. Both nicotine-containing and nicotine-free fluids induced an increased ROS production after 24 h, along with an increased Bax expression, followed by apoptosis occurrence after 48 h of exposure. The cytotoxicity exerted on HGFs by e-cigarettes fluids is not entirely ascribable to nicotine. Since the e-cigarettes are advertised as a safer alternative to traditional ones, especially for the possibility of "smoking" nicotine-free fluids, further studies are necessary to clarify the mechanism involved in the occurrence of cytotoxicity exerted by such compounds. Our results suggest a role for e-cigarette fluids in the pathogenesis of oral diseases, such as periodontitis.

Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

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Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-10-01

The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

Unilateral uveitis masquerade syndrome caused by diffuse large B-cell lymphoma diagnosed using multiparametric flow cytometry of the aqueous humor.

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Monsalvo, Silvia; Serrano, Cristina; Prieto, Elena; Fernández-Sanz, Guillermo; Puente, Maria-Camino; Rodriguez-Pinilla, Maria; Garcia Raso, Aranzazu; Llamas, Pilar; Cordoba, Raul

2017-07-01

The uveitis masquerade syndromes (UMS) are a group of ocular diseases that may mimic chronic intraocular inflammation. Many malignant entities such as non-Hodgkin's lymphomas may masquerade as uveitis. We report a case of an HIV-positive patient with masquerade syndrome presenting unilateral uveitis. 45-year-old Caucasian man with a diagnosis of diffuse large B-cell lymphoma (DLBCL). The patient was diagnosed by a biopsy of an abdominal mass which showed fragments of gastric mucosa with diffuse growth of neoplastic cells. At diagnosis, the patient suffered from unilateral blurring of vision and a sudden decrease of left-eye visual acuity. A slit-lamp examination of the left eye revealed a diagnosis of anterior uveitis. The patient exhibited no signs of posterior uveitis. An anterior-chamber paracentesis was performed and analyzed by multiparameter flow cytometry (MFC), showing cells CD45, CD19, CD20, CD22, and CD38 positives, and moderate expression of CD10 with kappa light chain restriction, showing a monoclonal B-cell population. The patient received CHOP-R with intrathecal methotrexate followed by consolidation high dose methotrexate obtaining a complete response which is ongoing. Differential diagnosis between chronic uveitis and ocular lymphoma may be challenging. We advocate anterior-chamber paracentesis in cases of refractory uveitis in patients with hematologic malignancies. © 2016 International Clinical Cytometry Society. © 2016 International Clinical Cytometry Society.

The future of cytotoxic therapy: selective cytotoxicity based on biology is the key

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Bono, Johann S de; Tolcher, Anthony W; Rowinsky, Eric K

2003-01-01

Although mortality from breast cancer is decreasing, 15% or more of all patients ultimately develop incurable metastatic disease. It is hoped that new classes of target-based cytotoxic therapeutics will significantly improve the outcome for these patients. Many of these novel agents have displayed cytotoxic activity in preclinical and clinical evaluations, with little toxicity. Such preferential cytotoxicity against malignant tissues will remain tantamount to the Holy Grail in oncologic therapeutics because this portends improved patient tolerance and overall quality of life, and the capacity to deliver combination therapy. Combinations of such rationally designed target-based therapies are likely to be increasingly important in treating patients with breast carcinoma. The anticancer efficacy of these agents will, however, remain dependent on the involvement of the targets of these agents in the biology of the individual patient's disease. Results of DNA microarray analyses have raised high hopes that the analyses of RNA expression levels can successfully predict patient prognosis, and indicate that the ability to rapidly 'fingerprint' the oncogenic profile of a patient's tumor is now possible. It is hoped that these studies will support the identification of the molecules driving a tumor's growth, and the selection of the appropriate combination of targeted agents in the near future

Cytotoxic effects of local anesthesia through lidocaine/ropivacaine on human melanoma cell lines

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Ding-Kun Kang

Full Text Available Abstract Background: Local anesthetics (LAs are generally considered as safe, but cytotoxicity has been reported for several local anesthetics used in humans, which is not well investigated. In the present study, the cytotoxicity of lidocaine, ropivacaine and the combination of lidocaine and ropivacaine were evaluated on human melanoma cell lines. Melphalan, a nitrogen mustard alkylating agent, was used as a control agent for comparison of cytotoxic activity. Methods: Melanoma cell lines, A375 and Hs294T, were exposed to 1 h to different concentrations of above agents. Cell-viability after exposure was determined by flow cytometry. Results: Investigated LAs showed detrimental cytotoxicity on studied melanoma cell lines in time- (p < 0.001, concentration- (p < 0.001, and agent dependant. In both A375 and Hs294T cell lines, minimum cell viability rates were found after 72 h of exposure to these agents. Lidocaine 2% caused a reduction of vital cells to 10% ± 2% and 14% ± 2% in A375 and Hs294T, respectively after 72 h of exposure. Ropivacaine 0.75% after 72 h reduced viable cells to 15% ± 3% and 25% ± 3% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to the combination was 10% ± 2% and 18% ± 2% in A375 and Hs294T, respectively. Minimum cell viability after 72 h exposure to melphalan was 8% ± 1% and 12% ± 2%, in A375 and Hs294T, respectively. Conclusion: LAs have cytotoxic activity on human melanoma cell lines in a time-, concentration- and agent-dependant manner. Apoptosis in the cell lines was mediated through activity of caspases-3 and caspases-8.

Panel development for multicolor flow-cytometry testing of proliferation and immunophenotype in hMSCs.

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Bradford, Jolene A; Clarke, Scott T

2011-01-01

Adult human mesenchymal stem cells (hMSC) are rare fibroblast-like cells capable of differentiation into a variety of cell tissues which include bone, cartilage, muscle, ligament, tendon, and adipose. Normal adult bone marrow and adipose tissue are the most common sources of these cells. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSC for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; and specific surface antigen expression. Direct measurement of proliferation combined with simultaneous detection of the ISCT-consensus immunophenotypic profile provides data that is used to determine the differentiation status and health of the cells. Flow cytometry provides a powerful technology that is routinely used to simultaneously and rapidly measure multiple parameters in a single sample. This chapter describes a flow cytometric panel for the simultaneous detection of immunophenotypic profile, proliferative capacity, and DNA content measurement in hMSC. Because a relatively small number of cells are needed with this approach, measurements can be made with minimal impact on expansion potential. The ability to assess antigen expression and proliferative status enables the investigator to make informed decisions on expansion and harvesting.

Relationship between conventional culture and flow cytometry for the diagnosis of urinary tract infection.

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García-Coca, Marta; Gadea, Ignacio; Esteban, Jaime

2017-06-01

Urine culture is the gold standard for the diagnosis of urinary tract infections (UTI). The use of flow cytometry analyzers (FCA) prior to culture allows for the quantification and recognition of cell components in urine to be automated and makes it possible to relate these data to the urine pathogens subsequently identified in cultures. Urine samples were assessed with the Sysmex UF-1000i analyzer. Those that met the criteria for culture (> 25 leukocytes/μL or > 385 bacteria/μL) were subjected to quantitative urine culture on chromogenic agar. Counts of red blood cells (RBC), white blood cells (WBC), epithelial cells (EC), and the kind of microorganisms identified in cultures were evaluated. A total of 17,483 samples were processed by FCA. Of these, 9057 met the criteria for culture. Urine cultures were reduced by 48.2%. The most common urine pathogen was Escherichia coli (60.3%). Negative urine cultures were significantly (p flow cytometer for screening urine samples allows for a reduction in the number of urine cultures. WBC values correlate well with the main urine pathogens related to UTI. The results observed for Enterococcus spp. suggest a low impact of these pathogens as a cause of UTI.

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

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van Andel, Esther; de Bus, Ian; Tijhaar, Edwin J; Smulders, Maarten M J; Savelkoul, Huub F J; Zuilhof, Han

2017-11-08

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Combining Cell Type-Restricted Adenoviral Targeting with Immunostaining and Flow Cytometry to Identify Cells-of-Origin of Lung Cancer.

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Best, Sarah A; Kersbergen, Ariena; Asselin-Labat, Marie-Liesse; Sutherland, Kate D

2018-01-01

Lung cancers display considerable intertumoral heterogeneity, leading to the classification of distinct tumor subtypes. Our understanding of the genetic aberrations that underlie tumor subtypes has been greatly enhanced by recent genomic sequencing studies and state-of-the-art gene targeting technologies, highlighting evidence that distinct lung cancer subtypes may be derived from different "cells-of-origin". Here, we describe the intra-tracheal delivery of cell type-restricted Ad5-Cre viruses into the lungs of adult mice, combined with immunohistochemical and flow cytometry strategies for the detection of lung cancer-initiating cells in vivo.

Hydrostatic Pressure Enhances Vital Staining with Carboxyfluorescein or Carboxydichlorofluorescein in Saccharomyces cerevisiae: Efficient Detection of Labeled Yeasts by Flow Cytometry

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Abe, Fumiyoshi

1998-01-01

The extent of intracellular accumulation of the fluorescent dye carboxyfluorescein or carboxydichlorofluorescein (CDCF) in Saccharomyces cerevisiae was found to be increased 5- to 10-fold under a nonlethal hydrostatic pressure of 30 to 50 MPa. This observation was confirmed by analysis of individual labeled cells by flow cytometry. The pressure-induced enhancement of staining with CDCF required d-glucose and was markedly inhibited by 2-deoxy-d-glucose, suggesting that glucose metabolism has a role in the process. PMID:9501452

La citometría de flujo en el estudio de tumores mediastinales ricos en elementos linfoides Flow cytometry for the study of mediastinal tumors with abundant lymphoid elements

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Gerardo Vides Almonacid

2008-02-01

Full Text Available El mediastino anterior es un sitio frecuente de localización de tumores ricos en elementos linfoides. La identificación correcta de cada entidad es de importancia en el tratamiento de los pacientes. En ocasiones puede plantearse el diagnóstico diferencial entre timoma y linfoma linfoblástico con fenotipo de precursor T (LLB-T. La Citometría de Flujo (CF es una técnica complementaria útil para estos tumores de la cual se obtiene información cualitativa y cuantitativa. Revisamos 38 tumores mediastinales que tenían estudio de CF. Además comparamos los resultados de CF de timomas y tejido tímico normal con 42 casos de LLB-T de otras localizaciones anatómicas. De los 38 tumores mediastinales 6 eran lesiones benignas, 9 linfomas difusos de células grandes con fenotipo B (LDCG-B, 10 linfomas de Hodgkin (LH, 11 timomas y 2 LLB-T. En 24 casos la CF aportó información positiva, definiendo el inmunofenotipo de las células linfoides neoplásicas, o los linfocitos característicos que acompañan a los timomas. La CF en los 10 casos de LH y en 4 lesiones benignas permitió descartar otros tipos de linfoma (LDCG-B, LLB-T, etc.. Las marcaciones para CD3, CD4 y CD8 fueron las más útiles para el diagnóstico diferencial entre timomas y LLB-T. En conclusión, la CF es una técnica complementaria de utilidad que aporta información en lesiones mediastinales de manera rápida, requiriendo cantidades pequeñas de material, tanto para el diagnóstico inicial como para el monitoreo de estas enfermedades.The anterior mediastinum is a common site of tumors with abundant lymphoid elements. Flow cytometry is a useful complementary technique to analyze this type of tumors, which provides qualitative and quantitative information. A differential diagnosis can be sometimes made between thymoma and precursor T-lymphoblastic lymphoma (T-LBL. Correct identification is of utmost importance for patient treatment. A total of 38 mediastinal tumors were analyzed, and

Taraxacum officinale induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

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Koo, Hyun-Na; Hong, Seung-Heon; Song, Bong-Keun; Kim, Cheorl-Ho; Yoo, Young-Hyun; Kim, Hyung-Min

2004-01-16

Taraxacum officinale (TO) has been frequently used as a remedy for women's disease (e.g. breast and uterus cancer) and disorders of the liver and gallbladder. Several earlier studies have indicated that TO exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we investigated the effect of TO on the cytotoxicity and production of cytokines in human hepatoma cell line, Hep G2. Our results show that TO decreased the cell viability by 26%, and significantly increased the tumor necrosis factor (TNF)-alpha and interleukin (IL)-1alpha production compared with media control (about 1.6-fold for TNF-alpha, and 2.4-fold for IL-1alpha, P < 0.05). Also, TO strongly induced apoptosis of Hep G2 cells as determined by flow cytometry. Increased amounts of TNF-alpha and IL-1alpha contributed to TO-induced apoptosis. Anti-TNF-alpha and IL-1alpha antibodies almost abolished it. These results suggest that TO induces cytotoxicity through TNF-alpha and IL-1alpha secretion in Hep G2 cells.

FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

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Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

2015-11-01

In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

Synthesis and cytotoxicity evaluation of thiosemicarbazones and their thiazole derivatives

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Saulo Feheiberg Pinto Braga

Full Text Available ABSTRACT The aims of this study were to synthesize a series of thiosemicarbazones and their thiazole derivatives, to investigate their cytotoxic activity against three human cancers and normal (Vero cells cell lines, and to evaluate the pro-apoptotic potential of the most active compounds. Materials and Methods: The thiosemicarbazones were obtained by reacting an aromatic aldehyde with thiosemicarbazide (yield 71-96%, which were subjected to a cyclization with α-bromoacetophenone to yield the required thiazole heterocycles (yield 63-100%. All the synthesized compounds were screened at 50 µM concentration against three cell lines representing HL60 (promyelocytic leukemia, Jurkat (acute lymphoblastic leukemia, and MCF-7 (breast cancer. The pro-apoptotic effect was measured by flow cytometry as the percentage of cells with hypodiploid DNA. Results: Three thiazole compounds showed activity against at least one tumor cell line (IC50 = 43-76 µM and low cytotoxicity against Vero cells (IC50 > 100 M. The most active compound of this series induced 91% and 51% DNA fragmentation in HL60 and MCF-7 cell lines, respectively, suggesting that this compound triggered apoptosis in these cells. Conclusion: Among the synthesized compounds, one in particular was found to exert antiproliferative and pro-apoptotic activity on tumor cells and can be considered promising as a lead molecule for the design of new analogues with improved activity.

Chip-Based Cytometry Illuminated by a Blade-Shape Continuous Light for Multispectral Detection

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Shi-Wei Lin

2016-08-01

Full Text Available A high performance diascopic illumination configuration is presented for the simultaneous detection of cells and particles with different sizes and different fluorescence labels in a microchannel. In the proposed approach, the cells/particles are illuminated by an objective-type dark-field condenser equipped with a low-cost tungsten light source and are then characterized by extracting the side-scatter, absorbance, and fluorescence signals from the spectra obtained by a ultraviolet-visible-near infrared (UV-Vis-NIR spectrometer. A modified computation model is adopted to improve the capability for discriminating more fluorescence dyes simultaneously. The feasibility of the proposed detection configuration is demonstrated by counting and classifying a mixed sample of green, red, and crimson fluorescent-labeled particles and non-labeled particles with various dimensions. The suitability of the proposed system for real-world cytometry applications is then evaluated by classifying a mixed bio-sample comprising of gastric epithelial (AGS cells stained with Trypan-blue and Erythrosin-bluish dye, respectively. The results show that the cytometer enables the efficient detection, identification, and classification of mixed bio-samples without the need for spatial filters or delicate optical components. Consequently, the proposed system has significant potential for high-performance micro-flow cytometry applications.

Flow cytometry in the diagnosis of myelodysplastic syndromes (MDS) and the value of myeloid nuclear differentiation antigen (MNDA).

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Bellos, Frauke; Kern, Wolfgang

2014-09-25

Background: Confirming diagnosis of myelodysplastic syndromes (MDS) is often challenging. Standard diagnostic methods are cytomorphology (CM) and cytogenetics (CG). Multiparameter flow cytometry (MFC) is upcoming in MDS diagnostic work up, comparability and investigator experience are critical. Myeloid nuclear differentiation antigen (MNDA) in myelomonocytic cells might be expressed more weakly in patients with MDS. The analysis of MNDA may thus improve diagnostic capabilities of MFC in MDS. Methods: Staining methods and antibody combinations for MFC in MDS are outlined, giving details for interpretation of results in regard to dyspoiesis. MFC results are correlated with CM and CG and with survival data. Use of myeloid nuclear differentiation antigen (MNDA) in MDS diagnostics was evaluated in 239 patients with MDS, AML, other cytopenic conditions and in 30 negative controls. Results: Strong correlation between findings in CM and MFC was found; MFC results correlated well with those of CG. Patients with higher grades of dysplasia in MFC had shorter overall survival. Percentages of granulocytes and monocytes with diminished MNDA expression (%dimG, %dimM) were higher in patients with MDS and AML. Mean fluorescence intensity (MFI) of MNDA in monocytes was lower in MDS and AML. Cut-off values for %dimG (12%) and %dimM (22%) as well as for MFI in monocytes (72) were defined discriminating between MDS and non-MDS. Conclusion: MFC adds significant information on dyspoiesis in the diagnostic work up for MDS and provides prognostic information. MNDA expression can be assessed by MFC and may facilitate evaluation of dyspoiesis when added to MDS MFC panels. © 2014 Clinical Cytometry Society. Copyright © 2014 Clinical Cytometry Society.

Avoiding false positive antigen detection by flow cytometry on blood cell derived microparticles: the importance of an appropriate negative control.

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Emerence Crompot

Full Text Available Microparticles (MPs, also called microvesicles (MVs are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but there is no consensus on the appropriate negative control to use that can lead to false positive results.We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets. Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM and Scanning Electron microscopy (SEM.Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins.We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.

Flow cytometric characterization of cerebrospinal fluid cells.

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de Graaf, Marieke T; de Jongste, Arjen H C; Kraan, Jaco; Boonstra, Joke G; Sillevis Smitt, Peter A E; Gratama, Jan W

2011-09-01

Flow cytometry facilitates the detection of a large spectrum of cellular characteristics on a per cell basis, determination of absolute cell numbers and detection of rare events with high sensitivity and specificity. White blood cell (WBC) counts in cerebrospinal fluid (CSF) are important for the diagnosis of many neurological disorders. WBC counting and differential can be performed by microscopy, hematology analyzers, or flow cytometry. Flow cytometry of CSF is increasingly being considered as the method of choice in patients suspected of leptomeningeal localization of hematological malignancies. Additionally, in several neuroinflammatory diseases such as multiple sclerosis and paraneoplastic neurological syndromes, flow cytometry is commonly performed to obtain insight into the immunopathogenesis of these diseases. Technically, the low cellularity of CSF samples, combined with the rapidly declining WBC viability, makes CSF flow cytometry challenging. Comparison of flow cytometry with microscopic and molecular techniques shows that each technique has its own advantages and is ideally combined. We expect that increasing the number of flow cytometric parameters that can be simultaneously studied within one sample, will further refine the information on CSF cell subsets in low-cellular CSF samples and enable to define cell populations more accurately. Copyright © 2011 International Clinical Cytometry Society.

Nuclear DNA content of the hybrid plant pathogen Phytophthora andina determined by flow cytometry.

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Wang, Jianan; Presser, Jackson W; Goss, Erica M

2016-09-01

Phytophthora andina is a heterothallic plant pathogen of Andean solanaceous hosts and is an interspecific hybrid of P. infestans and an unknown Phytophthora species. The objective of this study was to estimate the nuclear DNA content of isolates in three clonal lineages of P. andina relative to P. infestans Twelve isolates of P. andina and six isolates of P. infestans were measured for nuclear DNA content by propidium iodide-stained flow cytometry. We found that the DNA content of P. andina was similar but slightly smaller, on average, than that of our sample of P. infestans isolates. This is consistent with P. andina being a homoploid hybrid rather than allopolyploid hybrid. Nuclear DNA content was more variable among a smaller sample of P. infestans isolates, including a putative triploid isolate from Mexico, but small differences in nuclear DNA content were also observed among P. andina isolates. Both species appear to be able to tolerate significant variation in genome size. © 2016 by The Mycological Society of America.

Analyse de données de cytometrie de flux pour un grand nombre d'échantillons

OpenAIRE

Chen , Xiaoyi

2015-01-01

In the course of my Ph.D. work, I have developed and applied two new computational approaches for automatic identification of cell populations in multi-parameter flow cytometry across a large number of samples. Both approaches were motivated and taken by the LabEX "Milieu Intérieur" study (hereafter MI study). In this project, ten 8-color flow cytometry panels were standardized for assessment of the major and minor cell populations present in peripheral whole blood, and data were collected an...

Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death.

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Movafegh, Bahareh; Jalal, Razieh; Mohammadi, Zobeideh; Aldaghi, Seyyede Araste

2018-04-11

Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide-acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicin-induced cell death. Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24 h combined treatment of cells with doxorubicin (0.5 μM) and poly-L-arginine (1 μg ml-1) caused a small increase in doxorubicin-induced apoptosis and significant elevated necrosis in DU145 cells as compared to each agent alone. Conlusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferation-inducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Stress response assessment of Lactobacillus sakei strains selected as potential autochthonous starter cultures by flow cytometry and nucleic acid double-staining analyses.

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Bonomo, M G; Milella, L; Martelli, G; Salzano, G

2013-09-01

The aim of this study was to apply the flow cytometry to Lactobacillus sakei strains, selected as potential autochthonous starters, to investigate dynamics and physiological heterogeneity of microbial behaviour under different stress conditions. A simultaneous nucleic acid double-staining assay was applied to discriminate cell populations in different physiological states after exposure to heat (50 and 55°C) and acid (pH 2·5 and 3·0) stresses. Alive cells with intact membranes, damaged cells still alive but with injured membranes, so with even a recovery ability, and dead cells with a permanent membrane damage were differentiated with a significant increase in damaged cells after stronger stress treatments. The existence and characteristics of subpopulations displaying heterogeneity in particular conditions are highly relevant, because specific subpopulations may show improved survival, changes and dynamics under stress conditions. This assay has potential for physiological research on lactic acid bacteria and for application in the food industry. The assessment of intermediate physiological states in Lb. sakei strains with recovery possibility could be an important criterion for application of potential starter cultures. Application of flow cytometry and characterization of sorted subpopulations may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations. © 2013 The Society for Applied Microbiology.

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

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Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.

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Burel, Julie G; Qian, Yu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Zapardiel-Gonzalo, Jose; Taplitz, Randy; Gilman, Robert H; Saito, Mayuko; de Silva, Aruna D; Vijayanand, Pandurangan; Scheuermann, Richard H; Sette, Alessandro; Peters, Bjoern

2017-02-15

In the context of large-scale human system immunology studies, controlling for technical and biological variability is crucial to ensure that experimental data support research conclusions. In this study, we report on a universal workflow to evaluate both technical and biological variation in multiparameter flow cytometry, applied to the development of a 10-color panel to identify all major cell populations and T cell subsets in cryopreserved PBMC. Replicate runs from a control donation and comparison of different gating strategies assessed the technical variability associated with each cell population and permitted the calculation of a quality control score. Applying our panel to a large collection of PBMC samples, we found that most cell populations showed low intraindividual variability over time. In contrast, certain subpopulations such as CD56 T cells and Temra CD4 T cells were associated with high interindividual variability. Age but not gender had a significant effect on the frequency of several populations, with a drastic decrease in naive T cells observed in older donors. Ethnicity also influenced a significant proportion of immune cell population frequencies, emphasizing the need to account for these covariates in immune profiling studies. We also exemplify the usefulness of our workflow by identifying a novel cell-subset signature of latent tuberculosis infection. Thus, our study provides a universal workflow to establish and evaluate any flow cytometry panel in systems immunology studies. Copyright © 2017 by The American Association of Immunologists, Inc.

Flow cytometry of human primary epidermal and follicular keratinocytes.

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Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

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

DEFF Research Database (Denmark)

Langerhuus, Sine Nygaard; Ingvartsen, Klaus Lønne; Bennedsgaard, Torben Werner

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 identifica......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...... 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...... 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...

Detection of T and B cells specific complement-fixing alloantibodies using flow cytometry: A diagnostic approach for a resource limited laboratory

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Dharmendra Jain

2017-01-01

Conclusions: We postulate that this method incorporates most of the features of all the available modalities (i.e., National Institute of Health-complement dependent lymphocytotoxicity, FCXM, cytotoxic FCXM and C4d-flowPRA yet cost-effective and best suited for resource-limited laboratory/ies which is a common scenario in developing countries.

Cytometry of mammalian sperm

Energy Technology Data Exchange (ETDEWEB)

Gledhill, B.L.

1983-10-11

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples.

Cytometry of mammalian sperm

International Nuclear Information System (INIS)

Gledhill, B.L.

1983-01-01

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. The accessibility of male cells makes them well suited for analytical cytology. We might automate the process of determining sperm morphology but should not do so solely for increased speed. Rather, richer tangible benefits will derive from cytometric evaluation through increased sensitivity, reduced subjectivity, standardization between investigators and laboratories, enhanced archival systems, and the benefits of easily exchanged standardized data. Inroads on the standardization of assays for motility and functional integrity are being made. Flow cytometric analysis of total DNA content of individual sperm is an insensitive means to detect exposure to reproductive toxins because of the small size and low frequency of the DNA content errors. Flow cytometry can be applied to determine the proportions of X- and Y-sperm in semen samples

In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom.

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Stephanie Stransky

2018-04-01

Full Text Available Envenomation by the bushmaster snake Lachesis muta muta is considered severe, characterized by local effects including necrosis, the main cause of permanent disability. However, cellular mechanisms related to cell death and tissue destruction, triggered by snake venoms, are poorly explored. The purpose of this study was to investigate the cytotoxic effect caused by L. m. muta venom in normal human keratinocytes and to identify the cellular processes involved in in cellulo envenomation. In order to investigate venom effect on different cell types, Alamar Blue assay was performed to quantify levels of cellular metabolism as a readout of cell viability. Apoptosis, necrosis and changes in mitochondrial membrane potential were evaluated by flow cytometry, while induction of autophagy was assessed by expression of GFP-LC3 and analyzed using fluorescence microscopy. The cytotoxic potential of the venom is shown by reduced cell viability in a concentration-dependent manner. It was also observed the sequential appearance of cells undergoing autophagy (by 6 hours, apoptosis and necrosis (12 and 24 hours. Morphologically, incubation with L. m. muta venom led to a significant cellular retraction and formation of cellular aggregates. These results indicate that L. m. muta venom is cytotoxic to normal human keratinocytes and other cell lines, and this toxicity involves the integration of distinct modes of cell death. Autophagy as a cell death mechanism, in addition to apoptosis and necrosis, can help to unravel cellular pathways and mechanisms triggered by the venom. Understanding the mechanisms that underlie cellular damage and tissue destruction will be useful in the development of alternative therapies against snakebites.

Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy.

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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.

The utility of flow cytometry in differentiating NK/T cell lymphoma from indolent and reactive NK cell proliferations.

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de Mel, Sanjay; Li, Jenny Bei; Abid, Muhammad Bilal; Tang, Tiffany; Tay, Hui Ming; Ting, Wen Chang; Poon, Li Mei; Chung, Tae Hoon; Mow, Benjamin; Tso, Allison; Ong, Kiat Hoe; Chng, Wee Joo; Liu, Te Chih

2018-01-01

The WHO defines three categories of NK cell malignancies; extra nodal NK/T cell lymphoma (NKTCL), aggressive NK cell leukemia, and the provisional entity chronic lymphoproliferative disorder of NK cells (CLPD-NK). Although the flow cytometric (FC) phenotype of CLPD-NK has been described, studies on FC phenotype of NKTCL are limited. To the best of our knowledge ours is the first study to compare the phenotype of NKTCL, CLPD-NK, reactive NK lymphocytosis (RNKL), and normal NK cells using eight color (8C) FC. Specimens analyzed using the Euroflow8C NK Lymphoproliferative Disorder (NKLPD) panel between 2011 and 2014 were identified from our database. All samples were analyzed on the FACSCantoII cytometer. NK cells were identified as CD45+, smCD3-, CD19-, CD56+ and normal T-cells served as internal controls. The majority of NKTCL were CD56 bright, CD16 dim, CD57-, and CD94+. CLPD-NK and RNKL were predominantly CD56+ or dim with positive expression of CD16 and CD57 and weak CD94 expression. Antigen based statistical analyses showed robust division of samples along the NKTCL/normal CD56 bright NK cell and CLPD-NK/RNKL/normal CD56 positive NK cell groups. It was concluded that FC can reliably distinguish NKTCL from CLPD-NK, normal NK cells of CD56+ phenotype, and RNKL. It was proposed that the typical phenotype for NKTCL is: CD56 bright, CD16 dim with positive CD2, CD7, CD94, HLADR, CD25, CD26, and absent CD57. This resembles the phenotype of the CD56 bright immunoregulatory subset of NK cells which we therefore hypothesize is the cell of origin of NKTCL. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Cetuximab Enhanced the Cytotoxic Activity of Immune Cells during Treatment of Colorectal Cancer

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Lin Wang

2017-11-01

Full Text Available Background/Aims: Cetuximab is a chimeric IgG1 monoclonal antibody which targets the extracellular domain of epidermal growth factor receptor. This antibody is widely used for colorectal cancer (CRC treatment but its influence on the immune system is incompletely understood. Methods: The immune influence of cetuximab therapy in CRC patients was investigated by analyzing peripheral blood mononuclear cells using flow cytometry. We undertook in vitro cytotoxicity and cytokine-profile assays to ascertain the immunomodulatory effect of cetuximab treatment. Results: The number of CD3+ T, CD8+ T, and natural killer (NK cells was increased significantly and T-regulatory cells reduced gradually after cetuximab treatment. Percentage of CD4+ T, natural killer T (NKT-like, invariant NKT, and dendritic cells was similar between baseline patients and cetuximab patients. Expression of CD137 on NK and CD8+ T cells was increased significantly after 4 weeks of cetuximab therapy. In vitro cetuximab treatment markedly increased expression of CD137 and CD107a on NK and CD8+ T cells. Cetuximab treatment promoted the cytotoxic activity of NK and CD8+ T cells against tumor cells. Conclusion: Cetuximab treatment promotes activation of the immune response but alleviates immunosuppression: this might be the underlying anti-CRC effect of cetuximab.

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds

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Nagarajan, Sounderya; Pioche-Durieu, Catherine; Tizei, Luiz H. G.; Fang, Chia-Yi; Bertrand, Jean-Rémi; Le Cam, Eric; Chang, Huan-Cheng; Treussart, François; Kociak, Mathieu

2016-06-01

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ~150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of

Extended flow cytometry characterization of normal bone marrow progenitor cells by simultaneous detection of aldehyde dehydrogenase and early hematopoietic antigens: implication for erythroid differentiation studies

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Pascariello Caterina

2008-05-01

Full Text Available Abstract Background Aldehyde dehydrogenase (ALDH is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH+ cells has been addressed by one single study (Gentry et al, 2007. The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii detection of ALDH+ cellular subsets in bone marrow from pure red cell aplasia patients. Results In normal bone marrow, we identified three populations of cells, namely ALDH+CD34+, ALDH-CD34+ and ALDH+CD34- (median percentages were 0.52, 0.53 and 0.57, respectively. As compared to ALDH-CD34+ cells, ALDH+CD34+ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH+CD34- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH+CD34- cells showed a CD71bright, CD105+, CD45- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a. Finally, ALDH+CD34- precursors were not detectable in patients with pure red cell aplasia (PRCA. Conclusion Our study, comparing surface antigen expression of

Application of the flow cytometry for determination of the amount of DNA in Yersinia pestis cells under the influence of serotonin (5-hydroxytryptamine)

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Korsukov, Vladimir N.; Shchukovskaya, Tatyana N.; Kravtsov, Alexander L.; Popov, Youri A.

2002-07-01

Using flow cytometry a low DNA content in inoculated Yersinia pestis EV cells have been shown at the beginning of culture in Hottinger broth pH 7.2. The dependence serotonin action of its concentration on DNA content have been demonstrated. Serotonin accelerated Yersinia pestis culture growth during cultivation in Hottinger broth pH 7.2 both at 28 degrees C and 37 degrees C at concentration of 10-5 M.

High-throughput isolation of giant viruses in liquid medium using automated flow cytometry and fluorescence staining.

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Jacques Yaacoub Bou Khalil

2016-01-01

Full Text Available The isolation of giant viruses using amoeba co-culture is tedious and fastidious. Recently, the procedure was successfully associated with a method that detects amoebal lysis on agar plates. However, the procedure remains time-consuming and is limited to protozoa growing on agar. We present here advances for the isolation of giant viruses. A high-throughput automated method based on flow cytometry and fluorescent staining was used to detect the presence of giant viruses in liquid medium. Development was carried out with the Acanthamoeba polyphaga strain widely used in past and current co-culture experiments. The proof of concept was validated with virus suspensions: artificially contaminated samples but also environmental samples from which viruses were previously isolated. After validating the technique, and fortuitously isolating a new Mimivirus, we automated the technique on 96-well plates and tested it on clinical and environmental samples using other protozoa. This allowed us to detect more than ten strains of previously known species of giant viruses and 7 new strains of a new virus lineage. This automated high-throughput method demonstrated significant time saving, and higher sensitivity than older techniques. It thus creates the means to isolate giant viruses at high speed.

Cytotoxicity of anthraquinones from the roots of Pentas schimperi towards multi-factorial drug-resistant cancer cells.

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Kuete, Victor; Donfack, Arno R Nanfack; Mbaveng, Armelle T; Zeino, Maen; Tane, Pierre; Efferth, Thomas

2015-08-01

Multidrug resistance in cancer represents a major problem in chemotherapy. The present study was designed to assess the cytotoxicity of anthraquinones from Pentas schimperi, namely damnacanthal (1), damnacanthol (2), 3-hydroxy-2-hydroxymethyl anthraquinone (3) and schimperiquinone B (4) against nine drug-sensitive and multidrug resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the above compounds, whilst caspase-Glo assay was used to detect the activation of caspases enzymes by compounds 1 and 2. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species were all analyzed via flow cytometry. Anthraquinones 1 and 2 displayed cytotoxic effects with IC50 values below 81 μM on all the nine tested cancer cell lines whilst 3 and 4 displayed selective activities. The recorded IC50 values for compounds 1 and 2 ranged from 3.12 μM and 12.18 μM (towards leukemia CCRF-CEM cells) and from 30.32 μM and 80.11 μM (towards gliobastoma U87MG.ΔEGFR cells) respectively, and from 0.20 μM (against CCRF-CEM cells) to 195.12 μM (against CEM/ADR5000 cells) for doxorubicin. Compounds 1 and 2 induced apoptosis in CCRF-CEM leukemia cells, mediated by the disruption of the MMP and increase in ROS production. Anthraquinones from Pentas schimperi and mostly 1 and 2 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug resistant cancers.

Individual Cell Based Traits Obtained by Scanning Flow-Cytometry Show Selection by Biotic and Abiotic Environmental Factors during a Phytoplankton Spring Bloom

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Pomati, Francesco; Kraft, Nathan J. B.; Posch, Thomas; Eugster, Bettina; Jokela, Jukka; Ibelings, Bas W.

2013-01-01

In ecology and evolution, the primary challenge in understanding the processes that shape biodiversity is to assess the relationship between the phenotypic traits of organisms and the environment. Here we tested for selection on physio-morphological traits measured by scanning flow-cytometry at the individual level in phytoplankton communities under a temporally changing biotic and abiotic environment. Our aim was to study how high-frequency temporal changes in the environment influence biodiversity dynamics in a natural community. We focused on a spring bloom in Lake Zurich (Switzerland), characterized by rapid changes in phytoplankton, water conditions, nutrients and grazing (mainly mediated by herbivore ciliates). We described bloom dynamics in terms of taxonomic and trait-based diversity and found that diversity dynamics of trait-based groups were more pronounced than those of identified phytoplankton taxa. We characterized the linkage between measured phytoplankton traits, abiotic environmental factors and abundance of the main grazers and observed weak but significant correlations between changing abiotic and biotic conditions and measured size-related and fluorescence-related traits. We tested for deviations in observed community-wide distributions of focal traits from random patterns and found evidence for both clustering and even spacing of traits, occurring sporadically over the time series. Patterns were consistent with environmental filtering and phenotypic divergence under herbivore pressure, respectively. Size-related traits showed significant even spacing during the peak of herbivore abundance, suggesting that morphology-related traits were under selection from grazing. Pigment distribution within cells and colonies appeared instead to be associated with acclimation to temperature and water chemistry. We found support for trade-offs among grazing resistance and environmental tolerance traits, as well as for substantial periods of dynamics in which

Evaluation of Trace Elements in Augmentation of Statin-Induced Cytotoxicity in Uremic Serum-Exposed Human Rhabdomyosarcoma Cells

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Hitoshi Uchiyama

2018-01-01

Full Text Available Patients with end-stage kidney disease (ESKD are at higher risk for rhabdomyolysis induced by statin than patients with normal kidney function. Previously, we showed that this increase in the severity of statin-induced rhabdomyolysis was partly due to uremic toxins. However, changes in the quantity of various trace elements in ESKD patients likely contribute as well. The purpose of this study is to determine the effect of trace elements on statin-induced toxicity in rhabdomyosarcoma cells exposed to uremic serum (US cells for a long time. Cell viability, apoptosis, mRNA expression, and intracellular trace elements were assessed by viability assays, flow cytometry, real-time RT-PCR, and ICP-MS, respectively. US cells exhibited greater simvastatin-induced cytotoxicity than cells long-time exposed with normal serum (NS cells (non-overlapping 95% confidence intervals. Intracellular levels of Mg, Mn, Cu, and Zn were significantly less in US cells compared to that in NS cells (p < 0.05 or 0.01. Pre-treatment with TPEN increased simvastatin-induced cytotoxicity and eliminated the distinction between both cells of simvastatin-induced cytotoxicity. These results suggest that Zn deficiencies may be involved in the increased risk for muscle complaints in ESKD patients. In conclusion, the increased severity of statin-induced rhabdomyolysis in ESKD patients may be partly due to trace elements deficiencies.

Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays.

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Fischer, Janine; Prosenc, Marc H; Wolff, Martin; Hort, Norbert; Willumeit, Regine; Feyerabend, Frank

2010-05-01

Magnesium (Mg) alloys are promising materials for the development of biodegradable implants. However, the current in vitro test procedures for cytotoxicity, cell viability and proliferation are not always suitable for this class of materials. In this paper we show that tetrazolium-salt-based assays, which are widely used in practice, are influenced by the corrosion products of Mg-based alloys. Corroded Mg converts tetrazolium salts to formazan, leading to a higher background and falsifying the results of cell viability. Tetrazolium-based assays are therefore not a useful tool for testing the cytotoxicity of Mg in static in vitro assays. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Optimization of the C11-BODIPY(581/591) dye for the determination of lipid oxidation in Chlamydomonas reinhardtii by flow cytometry.

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Cheloni, Giulia; Slaveykova, Vera I

2013-10-01

Lipid oxidation is a recognized end point for the study of oxidative stress and is an important parameter to describe the mode of micropollutant action on aquatic microorganisms. Therefore, the development of quick and reliable methodologies probing the oxidative stress and damage in living cells is highly sought. In the present proof-of-concept work, we examined the potential of the fluorescent dye C11-BODIPY(591/581) to probe lipid oxidation in the green microalga Chlamydomonas reinhardtii. C11-BODIPY(591/581) staining was combined with flow cytometry measurements to obtain multiparameter information on cellular features and oxidative stress damage within single cells. First, staining conditions were optimized by exploring the capability of the dye to stain algal cells under increasing cell and dye concentrations and different staining procedures. Then lipid oxidation in algae induced by short- and long-term exposures to the three metallic micropollutants, copper, mercury, and nanoparticulate copper oxide, and the two organic contaminants, diethyldithiocarbamate (DDC) and diuron was determined. In this work we pointed out C11-BODIPY(591/581) applicability in a wide range of exposure conditions, including studies of oxidation as a function of time and that it is suitable for in vivo measurements of lipid oxidation due to its high permeation and stability in cells and its low interference with algal autofluorescence. © 2013 International Society for Advancement of Cytometry. Copyright © 2013 International Society for Advancement of Cytometry.

Antibacterial Activity of Commercial Dentine Bonding Systems against E. faecalis–Flow Cytometry Study

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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.

A cationic amphiphilic peptide ABP-CM4 exhibits selective cytotoxicity against leukemia cells.

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Chen, Yu Qing; Min, Cui; Sang, Ming; Han, Yang Yang; Ma, Xiao; Xue, Xiao Qing; Zhang, Shuang Quan

2010-08-01

Some cationic antibacterial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells, which could provide a new class of anticancer drugs. In the present study, the anticancer activity of ABP-CM4, an antibacterial peptide from Bombyx mori, against leukemic cell lines THP-1, K562 and U937 was evaluated, and the cytotoxicity compared with the effects on non-cancerous mammalian cells, including peripheral blood mononuclear cells (PBMCs), HEK-293 and erythrocytes. ABP-CM4 reduced the number of viable cells of the leukemic cell lines after exposure for 24h. The reduction was concentration dependent, and the IC50 values ranged from 14 to 18 microM. Conversely, ABP-CM4, even at 120 microM, exhibited no cytotoxicity toward HEK-293 or PBMCs, indicating that there was no significant effect on these two types of non-cancer cells. ABP-CM4 at a concentration of 200 microM had no hemolytic activity on mammalian erythrocytes. Together, these results suggested a selective cytotoxicity in leukemia cells. Flow cytometry demonstrated that the binding activity of ABP-CM4 to leukemia cells was much higher than that to HEK-293 or PBMCs, and there was almost no binding to erythrocytes. FITC-labeled ABP-CM4 molecules were examined under a confocal microscope and found to be concentrated at the surface of leukemia cells and changes of the cell membrane were determined by a cell permeability assay, which led us to the conclusion that ABP-CM4 could act at the cell membrane for its anticancer activity on leukemia cells. Collectively, our results indicated that ABP-CM4 has the potential for development as a novel antileukemic agent. Copyright 2010 Elsevier Inc. All rights reserved.

Expression of the chondroitin sulphate proteoglycan molecular complex in six human melanoma xenograft lines studied by flow cytometry and immunohistochemistry.

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Nagelhus, T A; Rofstad, E K

1993-06-01

The expression of the chondroitin sulphate proteoglycan (CSP) molecular complex in six human melanoma xenograft lines (BEX-t, COX-t, HUX-t, ROX-t, SAX-t, WIX-t) was studied by flow cytometry and immunohistochemistry using the monoclonal antibodies 9.2.27, ME31.3, G7A5, and NKI.M6. The two methods and the four antibodies gave consistent results. The six melanoma lines could be divided into three distinct groups of two lines each; expression was high in the HUX-t and ROX-t lines and intermediate in the BEX-t and SAX-t lines, whereas the COX-t and WIX-t lines were negative. The mean number of epitopes per cell for 9.2.27 was approximately twice as high as for ME31.3, G7A5, and NKI.M6 and was estimated to range from 0.8 +/- 0.1 x 10(5) to 1.9 +/- 0.2 x 10(5) in the positive xenograft lines. The expression of the CSP complex was heterogeneous. The immunofluorescence histograms measured by flow cytometry were therefore broad for all tumour lines. A significant fraction of the HUX-t cells was negative or weakly stained. These cells appeared as clear negative patches in the immunohistochemical preparations. Moreover, most morphologically intact tumour cells adjacent to necrotic areas did not show significant expression of the CSP complex, irrespective of tumour line. These cells were probably hypoxic and thus resistant to radiation therapy. The expression of the CSP complex in the xenograft lines was similar to that reported for melanoma in man.

Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging of Hypochlorous Acid in Living Cells and Quantitative Application in Flow Cytometry.

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Zhan, Zixuan; Liu, Rui; Chai, Li; Li, Qiuyan; Zhang, Kexin; Lv, Yi

2017-09-05

Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system, and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective method for HClO detection in living systems, and most of fluorescent probes are mainly focused on cells imaging. Besides, accurate HClO quantitative information about individual cells in a large cell population is extremely important for understanding inflammation and cellular apoptosis as well. In our work, a turn-on fluorescent probe has been synthesized, which can selectively and sensitively detect HClO with fast response time. The probe is almost nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged C═N bonds which can undergo a nonradiative decay process in the excited state. Upon the addition of ClO - , the probe was oxidized to ring-opened fluorescent form and the fluorescence intensity was greatly enhanced. In live cell experiments, the probe was successfully applied to image exogenous ClO - in HeLa cells and endogenous HClO in RAW 264.7 macrophage cells. In particular, the quantitative information on exogenous and endogenous HClO can also be acquired in flow cytometry. Therefore, the probe not only can image exogenous and endogenous HClO but also provides a new and promising platform to quantitatively detect HClO in flow cytometry.

Ploidy levels among species in the 'Oxalis tuberosa alliance' as inferred by flow cytometry.

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Emshwiller, Eve

2002-06-01

The 'Oxalis tuberosa alliance' is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as 'oca'. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C-values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3.6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1.67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast-expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0.79 to 1.34 pg/2C.

European symposium on cytometry

International Nuclear Information System (INIS)

1987-01-01

This book of abstracts contains 59 contributions about cervical prescreening, expert systems, breast cancer, ploidy analysis, system and data evaluation, sampling, preparation and staining, image cytometry, general cytometry, cell kinetics with clinical applications. (AJ)

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

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

2015-03-01

The increasing industrial use of nanomaterials during the last decades poses a potential threat to the environment and in particular to organisms living in the aquatic environment. In the present study, the toxicity of zinc oxide nanoparticles (ZnO NPs) was investigated in Marine algae Chlorella vulgaris (C. vulgaris). High zinc dissociation from ZnONPs, releasing ionic zinc in seawater, is a potential route for zinc assimilation and ZnONPs toxicity. To examine the mechanism of toxicity, C. vulgaris were treated with 50mg/L, 100mg/L, 200mg/L and 300 mg/L ZnO NPs for 24h and 72h. The detailed cytotoxicity assay showed a substantial reduction in the viability dependent on dose and exposure. Further, flow cytometry revealed the significant reduction in C. vulgaris viable cells to higher ZnO NPs. Significant reductions in LDH level were noted for ZnO NPs at 300 mg/L concentration. The activity of antioxidant enzyme superoxide dismutase (SOD) significantly increased in the C. vulgaris exposed to 200mg/L and 300 mg/L ZnO NPs. The content of non-enzymatic antioxidant glutathione (GSH) significantly decreased in the groups with a ZnO NPs concentration of higher than 100mg/L. The level of lipid peroxidation (LPO) was found to increase as the ZnO NPs dose increased. The FT-IR analyses suggested surface chemical interaction between nanoparticles and algal cells. The substantial morphological changes and cell wall damage were confirmed through microscopic analyses (FESEM and CM). Copyright © 2014 Elsevier Inc. All rights reserved.

Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (-)-deoxypodophyllotoxin.

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Masuda, Toshiya; Oyama, Yasuo; Yonemori, Shigetomo; Takeda, Yoshio; Yamazaki, Yuko; Mizuguchi, Shinichi; Nakata, Mami; Tanaka, Tomochika; Chikahisa, Lumi; Inaba, Yuzuru; Okada, Yoshihiko

2002-06-01

The cytotoxic activity of methanol extracts of leaves collected from 39 seashore plants in Iriomote Island, subtropical Japan was examined on human leukaemia cells (K562 cells) using a flow cytometer with two fluorescent probes, ethidium bromide and annexin V-FITC. Five extracts (10 microg/mL) from Hernandia nymphaeaefolia, Cerbera manghas, Pongamia pinnata, Morus australis var. glabra and Thespesia populnea greatly inhibited the growth of K562 cells. When the concentration was decreased to 1 microg/mL, only one extract from H. nymphaeaefolia still inhibited the cell growth. A cytotoxic compound was isolated from the leaves by bioassay-guided fractionation and was identified as (-)-deoxypodophyllotoxin (DPT). The fresh leaves of H. nymphaeaefolia contained a remarkably high amount of DPT (0.21 +/- 0.07% of fresh leaf weight), being clarified by a quantitative HPLC analysis. DPT at 70-80 pM started to inhibit the growth of K562 cells in an all-or-none fashion and at 100 pM or more it produced complete inhibition in all cases. Therefore, the slope of the dose-response curve was very steep. DPT at 100 pM or more decreased the cell viability to 50%-60% and increased the number of cells undergoing apoptosis (annexin V-positive cells). The results indicate that DPT contributes to the cytotoxic action of the extract from the leaves of H. nymphaeaefolia on K562 cells. Copyright 2002 John Wiley & Sons, Ltd.

Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

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Grandinetti, Giovanna; Smith, Adam E.; Reineke, Theresa M.

2012-01-01

The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(L-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T443, are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that not only could direct nucleus permeabilization be a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups as well as polymer length plays a role in polyplex size and charge in addition to protein expression efficiency and toxicity. PMID:22175236

PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells

International Nuclear Information System (INIS)

Seo, Kang-Sik; Hwang, Byung-Doo; Kim, Jong-Seok; Park, Ji-Hoon; Song, Kyoung-Sub; Yun, Eun-Jin; Park, Jong-Il; Kweon, Gi Ryang; Yoon, Wan-Hee; Lim, Kyu

2014-01-01

Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells. Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively. We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G 1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner. These results suggest that the synergy between PMA and apicularen A is involved by

The Means: Cytometry and Mass Spectrometry Converge in a Single Cell Deep Profiling Platform

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