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Sample records for flow cytometric test

  1. Ultrasensitive flow cytometric analyses

    SciTech Connect

    Jett, J.H.; Cram, L.S.; Keller, R.A.; Martin, J.C.; Saunders, G.C.; Sklar, L.A.; Steinkamp, J.A.

    1993-01-01

    New techniques and approaches to cellular analysis being developed at the Los Alamos National Flow Cytometry Resource can be divided into those that improve sensitivity and those that move the technology into new areas by refining existing approaches. An example of the first category is a flow cytometric system capable of measuring the phase shift of fluorescence emitted by fluorophors bound to cells is being assembled. This phase sensitive cytometer is be capable of quantifying fluorescence life time on a cell-by-cell basis as well as using the phase sensitive detection to separate fluorescence emissions that overlap spectrally but have different lifetimes. A Fourier transform flow cytometer capable of measuring the fluorescence emission spectrum of individual labeled cells at rates approaching several hundred per second is also in the new technology category. The current implementation is capable of resolving the visible region of the spectrum into 8 bands. With this instrument, it is possible to resolve the contributions of fluorophors with overlapping emission spectra and to determine the emission spectra of dyes such as calcium concentration indicators that are sensitive to the physiological environment. Flow cytometric techniques have been refined to the point that it is possible to detect individual fluorescent molecules in solution as they flow past a laser beam. This capability has lead to a rapid DNA sequencing project. The goal of the project is to develop a technique that is capable of sequencing long strands of DNA (40,000 kb) at a rate of between 100 and 1,000 bases per second.

  2. An on-bacterium flow cytometric immunoassay for protein quantification.

    PubMed

    Lan, Wen-Jun; Lan, Wei; Wang, Hai-Yan; Yan, Lei; Wang, Zhe-Li

    2013-09-01

    The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein. PMID:23739299

  3. Flow Cytometric Findings in Hemophagocytic Lymphohistiocytosis

    PubMed Central

    McCall, Chad M.; Mudali, Shiyama; Arceci, Robert J.; Small, Donald; Fuller, Shirley; Gocke, Christopher D.; Vuica-Ross, Milena; Burns, Kathleen H.; Borowitz, Michael J.; Duffield, Amy S.

    2016-01-01

    Hemophagocytic lymphohistiocytosis (HLH) is an often fatal hyperinflammatory syndrome. HLH may be inherited, but it more commonly arises secondary to Epstein-Barr virus (EBV) or other infections, hematologic malignancies, or rheumatologic diseases. We identified 17 patients diagnosed with HLH who had flow cytometric analysis of peripheral blood or bone marrow performed at the time of diagnosis. Two patients had primary HLH, and the others had HLH secondary to EBV infection, hematologic malignancies, rheumatologic conditions, or tuberculosis. The marrow typically showed a reactive lymphocytosis and a marked left shift in myelopoiesis regardless of the etiology. Qualitative abnormalities were also found in several cases, including T-cell abnormalities in the majority of the EBV-associated HLH cases. While not specific, flow cytometric findings in HLH are different from the findings in uninvolved marrow samples, and care should be taken not to overinterpret immunophenotypic findings in these cases as indicative of a primary marrow disorder or lymphoma. PMID:22523218

  4. Flow cytometric determination of quantitative immunophenotypes

    NASA Astrophysics Data System (ADS)

    Redelman, Douglas; Ensign, Wayne; Roberts, Don

    2001-05-01

    Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

  5. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY TRIBUTYLTIN

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose dependent and time-dependent manner. he flow cytometric parameter axial light loss, a measu...

  6. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY OF TRIBUTYLIN

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributylin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythrleukemic cell (MELC) in a dose-dependent and time-dependent manner. he flow cytometric parameter axial light loss, a measure...

  7. Flow cytometric evaluation of multicystic dysplastic kidneys.

    PubMed

    Jung, W H; Peters, C A; Mandell, J; Vawter, G F; Retik, A B

    1990-08-01

    The most appropriate management of the multicystic dysplastic kidney remains controversial. At issue is the long-term risk of the development of malignancy in the multicystic dysplastic kidney. The association between renal dysplasia and neoplasia has not been confirmed, with only 6 cases of malignancy reported. Nephroblastomatosis, a probable precursor of Wilms tumor, has been found in 5 to 7% of the cases of multicystic dysplastic kidney when specifically sought. In an attempt to determine whether a relationship exists between renal dysplasia and neoplasia in terms of abnormalities of cellular deoxyribonucleic acid content we performed flow cytometric evaluation on 30 formalin fixed, paraffin embedded archival specimens of multicystic dysplastic kidneys. None of the kidneys had evidence of malignancy. Nuclear deoxyribonucleic acid ploidy studies were performed on single dissociated nuclei prepared by the technique of McLemore and associates and stained with propidium iodide. All specimens demonstrated a diploid pattern of deoxyribonucleic acid, including 3 specimens with nephroblastomatosis or extensive papillary growth, and no specimen demonstrated a tetraploid or aneuploid pattern. The mean G0/G1 fraction was 85.94% (standard deviation 4.59) and the mean S/G2/M fraction was 12.54% (standard deviation 4.72). These findings do not support or negate the potential for neoplasm associated with multicystic dysplastic kidney, since a diploid deoxyribonucleic acid pattern does not eliminate the possibility of the future development of malignancy. PMID:2374213

  8. Flow cytometric sexing of mammalian sperm.

    PubMed

    Garner, Duane L

    2006-03-15

    This review reexamines parameters needed for optimization of flow cytometric sexing mammalian sperm and updates the current status of sperm sexing for various species where this technology is currently being applied. Differences in DNA content have provided both a method to differentiate between these sex-determining gametes and a method to sort them that can be used for predetermining sex in mammals. Although the DNA content of all cells for each mammalian species is highly conserved, slight but measurable DNA content differences of sperm occur within species even among cattle breeds due to different sizes of Y-chromosomes. Most mammals produce flattened, oval-headed sperm that can be oriented within a sorter using hydrodynamic forces. Multiplying the percentage the difference in DNA content of the X- or Y-chromosome bearing sperm times the area of the flat profile of the sperm head gives a simple sorting index that suggests that bull and boar sperm are well suited for separation in a flow sorter. Successful sperm sexing of various species must take into account the relative susceptibilities of gametes to the stresses that occur during sexing. Sorting conditions must be optimized for each species to achieve acceptable sperm sexing efficiency, usually at 90% accuracy. In the commercial application of sperm sexing to cattle, fertility of sex-sorted bull sperm at 2 x 10(6)/dose remains at 70-80% of unsexed sperm at normal doses of 10 to 20 x 10(6) sperm. DNA content measurements have been used to identify the sex-chromosome bearing sperm populations with good accuracy in semen from at least 23 mammalian species, and normal-appearing offspring have been produced from sexed sperm of at least seven species. PMID:16242764

  9. Automated high-dimensional flow cytometric data analysis

    PubMed Central

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

    2009-01-01

    Flow cytometric analysis allows rapid single cell interrogation of surface and intracellular determinants by measuring fluorescence intensity of fluorophore-conjugated reagents. The availability of new platforms, allowing detection of increasing numbers of cell surface markers, has challenged the traditional technique of identifying cell populations by manual gating and resulted in a growing need for the development of automated, high-dimensional analytical methods. We present a direct multivariate finite mixture modeling approach, using skew and heavy-tailed distributions, to address the complexities of flow cytometric analysis and to deal with high-dimensional cytometric data without the need for projection or transformation. We demonstrate its ability to detect rare populations, to model robustly in the presence of outliers and skew, and to perform the critical task of matching cell populations across samples that enables downstream analysis. This advance will facilitate the application of flow cytometry to new, complex biological and clinical problems. PMID:19443687

  10. Flow cytometric immunofluorescence of rat anterior pituitary cells

    NASA Technical Reports Server (NTRS)

    Hatfield, J. Michael; Hymer, W. C.

    1985-01-01

    A flow cytometric immunofluorescence technique was developed for the quantification of growth hormone, prolactin, and luteinizing hormone producing cells. The procedure is based on indirect-immunofluorescence of intracellular hormone using an EPICS V cell sorter and can objectively count 50,000 cells in about 3 minutes. It can be used to study the dynamics of pituitary cell populations under various physiological and pharmacological conditions.

  11. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi,Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2011-07-05

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

  12. Flow cytometric detection method for DNA samples

    DOEpatents

    Nasarabadi, Shanavaz; Langlois, Richard G.; Venkateswaran, Kodumudi S.

    2006-08-01

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

  13. Flow cytometric detection of wild yeast in lager breweries.

    PubMed

    Jespersen, L; Lassen, S; Jakobsen, M

    1993-02-01

    A flow cytometric method for detection of wild yeast infections in breweries is reported. It is based on selective enrichment in Malt extract Yeast extract Glucose Peptone broth (MYGP) at 37 degrees C and in MYGP with 200 ppm CuSO4 at 25 degrees C, staining with a fluorochrome precursor and flow cytometry. In experiments with several types of wild yeast isolated from breweries and two different strains of lager yeast it has been possible to detect one wild yeast per 10(6) culture yeast after 48-72 h of incubation and, in some cases, after 24 h. PMID:8466805

  14. Commercially Available Antibodies to Human Tumour Necrosis Factor-α Tested for Cross-Reactivity with Ovine and Bovine Tumour Necrosis Factor-α using Flow Cytometric Assays

    PubMed Central

    Dernfalk, J; Waller, K Persson; Johannisson, A

    2004-01-01

    A thorough understanding of the immune system, including the role of different cytokines, during inflammatory diseases in ruminants could lead to the development of new diagnostic methods and treatments. Tumour necrosis factor-α (TNF-α) is an important cytokine in the onset of the inflammatory responses. Unfortunately, the number of studies on cytokines, like TNF-α, in ruminants is limited due to a lack of species-specific reagents. As cytokines have remained rather conserved during evolution, cross-reactivity between animal species may occur. Therefore, the aim of the present study was to investigate 5 commercially available antibodies against human TNF-α for their ability to cross-react with ovine and/or bovine TNF-α, using a bead-based flow cytometric method. Two of the antibody clones (Mab 11 and 6401.1111) showed cross reactivity with ovine recombinant TNF-α in concentrations above 2.5 ng/ml. However, none of the antibodies detected TNF-α in bovine milk, or serum containing known concentrations of bovine TNF-α, as earlier determined with ELISA. The results could be due to inability of the antibodies to cross-react between species, but quenching of the signal by matrix proteins might also have lowered the response. PMID:15535090

  15. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1986-01-01

    A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as bromodeoxyuridine (BrdU) is used as a probe for the measurement of BrdU uptake by the cells as a measure of DNA synthesis.

  16. Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.

    PubMed

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T

    2016-03-29

    Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET. PMID:26997269

  17. Uncovering aberrant mutant PKA function with flow cytometric FRET

    PubMed Central

    Lee, Shin-Rong; Sang, Lingjie; Yue, David T.

    2016-01-01

    SUMMARY Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPI). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell, FRET-based binding curves using a commercially-available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validate our binding assay against the gold-standard isothermal calorimetry (ITC), and use flow cytometric FRET to uncover the structural and functional effects of the Cushing syndrome-causing mutation (L206R) on PKA’s catalytic subunit. We discover that this mutation not only differentially affects PKAcat’s binding to its multiple partners, but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability and power of flow cytometric FRET. PMID:26997269

  18. Automated High-Dimensional Flow Cytometric Data Analysis

    NASA Astrophysics Data System (ADS)

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

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

  19. Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342 †

    PubMed Central

    Monger, Bruce C.; Landry, Michael R.

    1993-01-01

    We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 105 to 15 × 105 cells ml-1. PMID:16348898

  20. Flow Cytometric Analysis of Immune Cells Within Murine Aorta.

    PubMed

    Gjurich, Breanne N; Taghavie-Moghadam, Parésa L; Galkina, Elena V

    2015-01-01

    The immune system plays a critical role in the modulation of atherogenesis at all stages of the disease. However, there are many technical difficulties when studying the immune system within murine aortas. Common techniques such as PCR and immunohistochemistry have answered many questions about the presence of immune cells and mediators of inflammation within the aorta yet many questions remain unanswered due to the limitations of these techniques. On the other hand, cumulatively the flow cytometry approach has propelled the immunology field forward but it has been challenging to apply this technique to aortic tissues. Here, we describe the methodology to isolate and characterize the immune cells within the murine aorta and provide examples of functional assays for aortic leukocytes using flow cytometry. The method involves the harvesting and enzymatic digestion of the aorta, extracellular and intracellular protein staining, and a subsequent flow cytometric analysis. PMID:26445788

  1. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

    USGS Publications Warehouse

    Pascho, R.J.; Ongerth, J.E.

    2000-01-01

    The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated

  2. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation.

    PubMed

    Pascho, R J; Ongerth, J E

    2000-07-14

    The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 10(6) bacteria ml(-1) and the bacteria exposed to chlorine at 1 mg l(-1) for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 10(6) bacteria ml(-1) and exposed to 0.8 mg l(-1) free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p < or = 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 < or = 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 > or = 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation

  3. FLOW CYTOMETRIC ANALYSIS OF THE CELLULAR TOXICITY OF TRIBUTYLTIN (JOURNAL VERSION)

    EPA Science Inventory

    Flow cytometric and light/fluorescence microscopic analyses indicate that tributyltin (TBT) alters the plasma membrane/cytoplasm complex of the murine erythroleukemic cell (MELC) in a dose-dependent and time-dependent manner. The flow cytometric parameter axial light loss, a meas...

  4. Flow cytometric detection of pathogenic E. coli in food.

    PubMed

    Raybourne, R B

    2001-05-01

    E. coli O157:H7 is one of the more important food pathogens, andrapid, quantitative methods to evaluate foods for the presence of this pathogen are needed. This unit provides exactly that: a very much simplified flow cytometric assay for detection of E. coli O157:H7 in a well established vehicle of infection, ground beef. The method uses commercially available FITC-conjugated specific antibody to this bacterial serotype. Sample preparation and bacterial enrichment procedures are described. Direct and indirect approaches for quantification of the number of bacteria are given. A key feature of the assay is the reduction in time compared with plate-counting methods; the tradeoff is a slight reduction in sensitivity. Particularly useful is the simultaneous inclusion of a spiked sample to ensure a positive control. In addition, the unit provides hints on sorting the organisms if desired. PMID:18770690

  5. Oxidative product formation in irradiated neutrophils. A flow cytometric analysis

    SciTech Connect

    Wolber, R.A.; Duque, R.E.; Robinson, J.P.; Oberman, H.A.

    1987-03-01

    The effect of irradiation on neutrophil oxidative function was evaluated using a flow cytometric assay of intracellular hydrogen peroxide (H/sub 2/O/sub 2/) production. This assay quantitates the H/sub 2/O/sub 2/-dependent conversion of the nonfluorescent compound, 2'-7'-dichlorofluorescein (DCFH), into fluorescent 2'-7'-dichlorofluorescein (DCF) on a single-cell basis. Intracellular H/sub 2/O/sub 2/ production in response to stimulation with phorbol myristate acetate was not affected by neutrophil irradiation at doses up to 2500 rad. In addition, irradiation of intracellular DCFH and aqueous 2'-7'-dichlorofluorescein diacetate (DCFH-DA) resulted in DCF production, which suggested that oxidative molecules produced by aqueous radiolysis were detected by this assay. This study indicates that radiation doses of 1500 to 2500 rad, which are sufficient to prevent induction of graft-versus-host disease by transfused blood components, are not deleterious to neutrophil oxidative metabolism.

  6. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, Frank A.; Gray, Joe W.

    1988-01-01

    A method for the simultaneous flow cytometric measurement of the total DNA content and the level of DNA synthesis in normal and malignant cells is disclosed. The sensitivity of the method allows a study of cell cycle traverse rates for large scale cell populations as well as single cell measurements. A DNA stain such as propidium iodide or Hoechst 33258 is used as the probe for the measurement of total DNA content and a monoclonal antibody reactive with a DNA precursor such as halodeoxy-uridine (HdU), more specifically bromodeoxyuridine (BrdU) is used as a probe for the measurement of HdU or BrdU uptake by the cells as a measure of DNA synthesis.

  7. Flow cytometric cell-based assay to preselect antibody constructs for radionuclide conjugation.

    PubMed

    Ingargiola, M; Dittfeld, C; Runge, R; Zenker, M; Heldt, J-M; Steinbach, J; Cordes, N; Baumann, M; Kotzerke, J; Kunz-Schughart, L A

    2012-10-01

    Radiolabeled antibodies (Abs) are an attractive tool for targeting and delivering particle emitters for therapy or imaging applications. The labeling of Abs with metal radionuclides requires chelating agents and can cause loss of binding to their ligands. The aim of the present approach was to design an easy-handling flow cytometric cell-based assay to evaluate Ab-binding capacity of conjugates of the therapeutic Ab Cetuximab and to verify the most promising candidate in a competitive radioactive binding experiment. The final setup for flow cytometric assessment of cellular binding capacities of epidermal growth factor receptor (EGFR)/ErbB1-directed Ab conjugates is based on (a) the selection of a robust cell line model (b) the definition of nonsaturated staining concentrations for the unconjugated reference Ab Cetuximab plus implementation of a reasonable isotype control, and (c) the calculation of relative Ab affinities based on the flow cytometric data. Two (FaDu, SAS) out of the three cell lines with different total and cell surface expression levels of EGFR turned out to be adequate models but the application of one cell line was sufficient to estimate reduced binding capacities of conjugates relative to Cetuximab. Only 1/11 conjugate Abs exhibited a fluorescence signal comparable to unconjugated Cetuximab and was applied for radiolabeling with Yttrium-90. Unaltered binding affinity of this conjugate was proven in a competitive radioactive Ab-binding study. We conclude that the flow cytometric assay is reliable and that the relative binding capacity of Cetuximab is neither affected by covalent modification with CHX-A"-DTPA (N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl) propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N',N",N"-pentaacetic acid) with a final chelator-to-Ab ratio of 5 nor by subsequent radiolabeling. [(90)Y]Y-CHX-A"-DTPA-Cetuximab thus qualifies for preclinical treatment testing as a prerequisite for therapeutic application. PMID:22930585

  8. Flow cytometric detection of oxidative DNA damage in fish spermatozoa exposed to cadmium - Short communication.

    PubMed

    Nagy, Szabolcs; Kakasi, Balázs; Bercsényi, Miklós

    2016-03-01

    The aim of the present pilot study was to apply a flow cytometric assay, the so-called OxyDNA test, to determine the level of oxidative DNA damage in fish spermatozoa exposed to different concentrations (0.01-10,000 mg/L) of cadmium. Milt was collected from three randomly selected Prussian carp (Carassius auratus gibelio) males. Oxidative DNA damage was assessed with the OxyDNA kit and using flow cytometry. The ratio of OxyDNA-positive events increased significantly at higher cadmium concentrations. The results indicate that direct contact of fish spermatozoa with cadmium-polluted water initiates genotoxic damage. PMID:26919149

  9. The cytometric future: it ain't necessarily flow!

    PubMed

    Shapiro, Howard M

    2011-01-01

    Initial approaches to cytometry for classifying and characterizing cells were based on microscopy; it was necessary to collect relatively high-resolution images of cells because only a few specific reagents usable for cell identification were available. Although flow cytometry, now the dominant cytometric technology, typically utilizes lenses similar to microscope lenses for light collection, improved, more quantitative reagents allow the necessary information to be acquired in the form of whole-cell measurements of the intensities of light transmission, scattering, and/or fluorescence.Much of the cost and complexity of both automated microscopes and flow cytometers arises from the necessity for them to measure one cell at a time. Recent developments in digital camera technology now offer an alternative in which one or more low-magnification, low-resolution images are made of a wide field containing many cells, using inexpensive light-emitting diodes (LEDs) for illumination. Minimalist widefield imaging cytometers can provide a smaller, less complex, and substantially less expensive alternative to flow cytometry, critical in systems intended for in resource-poor areas. Minimalism is, likewise, a good philosophy in developing instrumentation and methodology for both clinical and large-scale research use; it simplifies quality assurance and compliance with regulatory requirements, as well as reduces capital outlays, material costs, and personnel training requirements. Also, importantly, it yields "greener" technology. PMID:21116998

  10. Reticulated platelets interfere with flow cytometric reticulocyte counts.

    PubMed

    Ivory, K; Sarria, B; Fairweather-Tait, S J; Hughes, D A

    2007-10-01

    As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis. PMID:17824916

  11. Flow cytometric analysis of circulating microparticles in plasma.

    PubMed

    Orozco, Aaron F; Lewis, Dorothy E

    2010-06-01

    Microparticles, which include exosomes, micro-vesicles, apoptotic bodies and apoptotic microparticles, are small (0.05 - 3 mum in diameter), membranous vesicles that can contain DNA, RNA, miRNA, intracellular proteins and express extracellular surface markers from the parental cells. They can be secreted from intracellular multivesicular bodies or released from the surface of blebbing membranes. Circulating microparticles are abundant in the plasma of normal individuals and can be derived from circulating blood cells such as platelets, red blood cells and leukocytes as well as from tissue sources, such as endothelial and placental tissues. Elevated levels of microparticles are associated with various diseases such as thrombosis (platelet microparticles), congestive heart failure (endothelial microparticles), breast cancer patients (leukocyte microparticles) and women with preeclampsia (syncytiotrophoblast microparticles). Although microparticles can be detected by microscopy, enzyme-linked immunoassays and functional assays, flow cytometry is the preferred method because of the ability to quantitate (fluorescent bead- or flow rate-based method) and because of polychromatic capabilities. However, standardization of pre-analytical and analytical modus operandi for isolating, enumerating and fluorescent labeling of microparticles remains a challenge. The primary focus of this article is to review the preliminary steps required to optimally study circulating in vivo microparticles which include: 1) centrifugation speed used, 2) quantitation of microparticles before antibody labeling, 3) levels of fluorescence intensity of antibody-labeled microparticles, 4) polychromatic flow cytometric analysis of microparticle sub-populations and 5) use of polyclonal antibodies designed for Western blotting for flow cytometry. These studies determine a roadmap to develop microparticles as biomarkers for a variety of conditions. PMID:20235276

  12. Detection of circulating immune complexes by Raji cell assay: comparison of flow cytometric and radiometric methods

    SciTech Connect

    Kingsmore, S.F.; Crockard, A.D.; Fay, A.C.; McNeill, T.A.; Roberts, S.D.; Thompson, J.M.

    1988-01-01

    Several flow cytometric methods for the measurement of circulating immune complexes (CIC) have recently become available. We report a Raji cell flow cytometric assay (FCMA) that uses aggregated human globulin (AHG) as primary calibrator. Technical advantages of the Raji cell flow cytometric assay are discussed, and its clinical usefulness is evaluated in a method comparison study with the widely used Raji cell immunoradiometric assay. FCMA is more precise and has greater analytic sensitivity for AHG. Diagnostic sensitivity by the flow cytometric method is superior in systemic lupus erythematosus (SLE), rheumatoid arthritis, and vasculitis patients: however, diagnostic specificity is similar for both assays, but the reference interval of FCMA is narrower. Significant correlations were found between CIC levels obtained with both methods in SLE, rheumatoid arthritis, and vasculitis patients and in longitudinal studies of two patients with cerebral SLE. The Raji cell FCMA is recommended for measurement of CIC levels to clinical laboratories with access to a flow cytometer.

  13. Carcinoma of the anal canal and flow cytometric DNA analysis.

    PubMed Central

    Scott, N. A.; Beart, R. W.; Weiland, L. H.; Cha, S. S.; Lieber, M. M.

    1989-01-01

    Using flow cytometric DNA analysis of paraffin embedded tissue, DNA histograms were successfully obtained from the anal cancers of 117 patients. DNA diploid patterns were given by 82 cancers (70%) and DNA non-diploid patterns by 35 cancers (30%): 15 DNA aneuploid, 20 DNA tetraploid. Well differentiated squamous cell cancers were mainly DNA diploid, while a larger proportion of poorly differentiated and small cell cancers were DNA non-diploid. The large majority of stage A cancers were DNA diploid. A greater proportion of tumours that had invaded through the anal sphincter or had lymph node metastases or distant spread were DNA non-diploid. Prognosis was slightly poorer for patients with DNA non-diploid cancers when compared to patients with DNA diploid tumours (P = 0.08) and significantly poorer for individuals with DNA aneuploid anal cancers (P = 0.037). However, in a multivariate analysis model, the DNA ploidy pattern of an anal cancer was not of independent prognostic significance alongside tumour histology and tumour stage. PMID:2803916

  14. Flow cytometric life cycle analysis in cellular radiation biology

    SciTech Connect

    Wood, J.C.S.

    1982-01-01

    Three approaches to flow cytometric histogram analysis were developed: (1) differential histogram analysis, (2) DNA histogram analysis, and (3) multiparameter data analysis. These techniques were applied to an important unresolved problem in radiation biology. The initial responses to irradiation of a mammalian cell which occur during the first two cell cycles following the irradiation are of considerable interest to the radiation biologist. During the first two post-irradiation cell cycles, cells which ultimately will survive repair radiation-induced damage, while some cells begin to express some of the radiation-induced nuclear and chomatin damage. Caffeine- and thymidine-treated, and untreated gamma-irradiated cell populations were studied with respect to the radiation-induced G2 delay, deficient DNA synthesis, and the appearance of cells with abnormal DNA contents. It is hypothesized that the measured deficiency in DNA synthesis observed in the first post-irradiation cell cycle may be a result of daughter cells from abnormal first post-irradiation mitoses.

  15. COMPARISON OF CELLULAR AND NUCLEAR FLOW CYTOMETRIC TECHNIQUES FOR DISCRIMINATING APOPTOTIC SUBPOPULATIONS

    EPA Science Inventory

    We compared cellular flow cytometric methods employing carboxyfluorescein (CF), Hoechst 33342, and Hoechst 33258 with a nuclear method in their ability to discriminate apoptotic subpopulations in rat thymocyte cultures exposed to dexamethasone r tributyltin. n the nuclear techniq...

  16. Flow cytometric analysis of crayfish haemocytes activated by lipopolysaccharides

    USGS Publications Warehouse

    Cardenas, W.; Dankert, J.R.; Jenkins, J.A.

    2004-01-01

    Lipopolysaccharides (LPS) from Gram-negative bacteria are strong stimulators of white river crayfish, Procambarus zonangulus, haemocytes in vitro. Following haemocyte treatment with LPS and with LPS from rough mutant R5 (LPS Rc) from Salmonella minnesota, flow cytometric analysis revealed a conspicuous and reproducible decrease in cell size as compared to control haemocytes. These LPS molecules also caused a reduction in haemocyte viability as assessed by flow cytometry with the fluorescent dyes calcein-AM and ethidium homodimer. The onset of cell size reduction was gradual and occurred prior to cell death. Haemocytes treated with LPS from S. minnesota without the Lipid A moiety (detoxified LPS) decreased in size without a reduction of viability. The action of LPS on crayfish haemocytes appeared to be related to the activation of the prophenoloxidase system because phenoloxidase (PO)-specific activity in the supernatants from control and detoxified LPS-treated cells was significantly lower than that from LPS and LPS-Rc treated cells (P < 0.05). Furthermore, addition of trypsin inhibitor to the LPS treatments caused noticeable delays in cell size and viability changes. These patterns of cellular activation by LPS formulations indicated that crayfish haemocytes react differently to the polysaccharide and lipid A moieties of LPS, where lipid A is cytotoxic and the polysaccharide portion is stimulatory. These effects concur with the general pattern of mammalian cell activation by LPS, thereby indicting commone innate immune recognition mechanisms to bacterial antigens between cells from mammals and invertebrates. These definitive molecular approaches used to verify and identify mechanisms of invertbrate haemocyte responses to LPS could be applied with other glycoconjugates, soluble mediators, or xenobiotic compounds.

  17. Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.

    PubMed

    Poujol, Fanny; Monneret, Guillaume; Friggeri, Arnaud; Rimmelé, Thomas; Malcus, Christophe; Poitevin-Later, Françoise; Pachot, Alexandre; Lepape, Alain; Textoris, Julien; Venet, Fabienne

    2014-12-15

    In clinical laboratories, the evaluation of lymphocyte proliferative response (lymphocyte transformation test-LTT) is routinely performed by the measurement of [(3)H]-thymidine uptake after stimulation. In this study we evaluated the performances of a recently developed non-radioactive test based on the detection by flow cytometry of 5-ethynyl-2'deoxyuridine (EdU) incorporation for the measurement of LTT in routine lab conditions. After definition of optimal protocol parameters, EdU incorporation test showed good repeatability and reproducibility. Moreover, this assay was flexible enough to fit important clinical laboratory constraints (delayed stimulation, low number of cells and delayed analysis after staining). Importantly, correlations between results obtained with EdU and [(3)H]-thymidine incorporation assays were excellent both in healthy volunteers and pediatric and septic patients. In particular, the two techniques identified patients presenting with altered LTT. Upon confirmation in a larger cohort of patients, EdU incorporation assay may be a relevant non-radioactive candidate for LLT in clinic. PMID:25450005

  18. Validation of a flow cytometric acridine orange micronuclei methodology in rats.

    PubMed

    Criswell, K A; Krishna, G; Zielinski, D; Urda, G A; Juneau, P; Bulera, S; Bleavins, M R

    2003-07-25

    Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to

  19. Quantifying Distribution of Flow Cytometric TCR-Vβ Usage with Economic Statistics

    PubMed Central

    van der Geest, Kornelis S. M.; Abdulahad, Wayel H.; Horst, Gerda; Lorencetti, Pedro G.; Bijzet, Johan; Arends, Suzanne; van der Heiden, Marieke; Buisman, Anne-Marie; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M. H.

    2015-01-01

    Measuring changes of the T cell receptor (TCR) repertoire is important to many fields of medicine. Flow cytometry is a popular technique to study the TCR repertoire, as it quickly provides insight into the TCR-Vβ usage among well-defined populations of T cells. However, the interpretation of the flow cytometric data remains difficult, and subtle TCR repertoire changes may go undetected. Here, we introduce a novel means for analyzing the flow cytometric data on TCR-Vβ usage. By applying economic statistics, we calculated the Gini-TCR skewing index from the flow cytometric TCR-Vβ analysis. The Gini-TCR skewing index, which is a direct measure of TCR-Vβ distribution among T cells, allowed us to track subtle changes of the TCR repertoire among distinct populations of T cells. Application of the Gini-TCR skewing index to the flow cytometric TCR-Vβ analysis will greatly help to gain better understanding of the TCR repertoire in health and disease. PMID:25923356

  20. Flow cytometric measurement of immunoglobulin E to natural latex proteins.

    PubMed Central

    Kwittken, P L; Pawlowski, N A; Sweinberg, S K; Douglas, S D; Campbell, D E

    1994-01-01

    Immediate hypersensitivity to natural latex (NL) occurs in sensitized individuals after repeated exposure to products or devices containing NL components. Since allergic reactions to NL proteins are quite frequent and may be quite serious, diagnostic assays are needed to identify individuals at risk. A number of latex proteins have been considered the major antigens, but they have been incompletely characterized. There is no standard material available for skin testing. In vitro diagnostic tests, such as the radioallergosorbent test (RAST), are time consuming and their sensitivity and specificity remain to be proven. We have developed a rapid microsphere-based, fluorescence-activated flow cytometry assay for the measurement of NL protein-specific human immunoglobulin E and have compared it with both the enzyme-linked immunosorbent assay and radioallergosorbent test methods. By using the total purified NL protein fraction isolated from raw ammoniated NL sap as the antigen, the flow cytometry assay was both sensitive and specific for the detection of NL protein-specific human immunoglobulin E in the sera of sensitized pediatric patients. PMID:7496945

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

    PubMed Central

    Tzircotis, George; Thorne, Rick F; Isacke, Clare M

    2004-01-01

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

  2. Flow cytometric determination of the photoinduced toxicity of anthracene to the green alga selenastrum capricornutum

    SciTech Connect

    Gala, W.R.; Giesy, J.P. . Dept. of Fisheries and Wildlife)

    1994-05-01

    Certain PAHs are photosensitizers and in the presence of solar radiation can cause toxicity to aquatic plants and animals. The photoinduced toxicity of anthracene to the green alga Selenastrum capricornutum was assessed by the use of flow cytometry to measure cell size, cellular chlorophyll concentration, and cell viability. Anthracene was slightly toxic in the absence of UV-A radiation. The detection of the direct toxicity of anthracene in this study at a concentration of 19 [mu]g/L anthracene resulted from the use of sensitive flow cytometric measures. There was a significant interaction between anthracene and UV-A radiation, which, in combination, caused significant toxic effects on Selenastrum capricornutum. The most sensitive flow cytometric measure of toxicity was the stress index (SI), which was predictive of longer term effects on cell growth. The 28-h EC50 and EC10 and for the SI for Selenastrum capricornutum were 16.1 and 8.3 [mu]g/L anthracene, respectively, at 125 [mu]W/cm[sup 2] UV-A. All combinations for anthracene and UV-A that inhibited algal growth also caused a significantly greater number of nonviable cells. The flow cytometric methods used in this study proved to be sensitive, predictive measures of the direct and photo-induced toxicity of anthracene and UV-A radiation to Selenastrum capricornutum.

  3. Intracellular Flow Cytometric Measurement of Extracellular Matrix Components in Porcine Intervertebral Disc Cells

    PubMed Central

    Flagler, Daniel J.; Huang, Chun-Yuh; Yuan, Tai-Yi; Lu, Zhongmin; Cheung, Herman S.; Gu, Wei Yong

    2009-01-01

    The objective of this study was to develop and demonstrate the utility of a novel method of evaluating intracellular levels of extracellular matrix (ECM) components in intervertebral disc (IVD) cells using flow cytometry. By using this method, this study discriminated between cell populations in porcine IVD and examined the response of IVD cells to monolayer cultures, a traditional method of cell expansion, by measuring phenotypic attributes of ECM component production. It was found that monolayer cultures affected collagen production of IVD cells while there were differences in collagen type II production between the cells isolated from the annulus fibrosus (AF) and nucleus pulposus (NP) regions of IVD. Size distributions of fresh and cultured cells were also presented while the relationships between cell size and intracellular collagen level revealed heterogeneous cell populations in AF and NP regions. Furthermore, this study showed that the intracellular collagen signals of IVD cells were significantly enhanced by the treatments of Brefeldin-A and ascorbic acid. This suggests that Brefeldin-A and ascorbic acid could be used to increase the sensitivity of flow cytometric analysis on intracellular collagen levels by maximizing collagen accumulation inside cells. Since a unique feature of the flow cytometric screening tool is the ability to discriminate between various cell populations in a single sample, the flow cytometric method developed in this study may have the potential to identify specific collagen-producing cell populations from tissues or cell cultures. PMID:20161070

  4. High speed flow cytometric separation of viable cells

    DOEpatents

    Sasaki, Dennis T.; Van den Engh, Gerrit J.; Buckie, Anne-Marie

    1995-01-01

    Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

  5. High speed flow cytometric separation of viable cells

    DOEpatents

    Sasaki, D.T.; Van den Engh, G.J.; Buckie, A.M.

    1995-11-14

    Hematopoietic cell populations are separated to provide cell sets and subsets as viable cells with high purity and high yields, based on the number of original cells present in the mixture. High-speed flow cytometry is employed using light characteristics of the cells to separate the cells, where high flow speeds are used to reduce the sorting time.

  6. Flow cytometric measurement of microparticles: pitfalls and protocol modifications.

    PubMed

    Shah, Mona D; Bergeron, Angela L; Dong, Jing-Fei; López, José A

    2008-08-01

    Upon activation, many cells shed components of their plasma membranes as microparticles. Depending on the methods of preparation and analyses, microparticle counts may vary significantly between laboratories, making data analyses and clinical correlations challenging. To assess how variations in sample preparation affect microparticle measurements, blood samples from 13 healthy, adult volunteers were labeled with Annexin V, cell-specific antibodies, and antibodies against tissue factor (TF). Data were acquired and analysed using an EPICS XL-MCL flow cytometer. Annexin V(+) monocyte-, platelet-, endothelial-, or erythrocyte-derived microparticles accounted for 10.4%, 38.5%, 43.8%, and 7.3% of the total number of microparticles (13.7 +/- 3.0 x 10(3)/ml of whole blood), respectively. A similar distribution of cell types was seen for TF(+) microparticles (6.3 +/- 2.6 x 10(3)/ml of whole blood). No statistical difference was noted in microparticle distribution using either 19- or 21-gauge needles. Elevated levels of platelet- and erythrocyte-derived microparticles were detected in heparin and PPACK-anticoagulated samples as compared to samples anticoagulated with ACD or sodium citrate (P < 0.05, student's t-test). Additional centrifugation was critical for removing platelet contamination, which significantly affected microparticle counts. Finally, Annexin V(+) and TF(+) microparticles were significantly reduced upon sample storage at low temperatures. Microparticle levels are significantly affected by variations in sample preparation and storage. These results illustrate the need to standardize assay protocols in order to obtain consistent measurements. Our studies further optimize sample preparation for microparticle detection. PMID:18791943

  7. Application and commercialization of flow cytometrically sex-sorted semen.

    PubMed

    Rath, D; Johnson, L A

    2008-07-01

    The current technology to sort X and Y chromosome bearing sperm population requires individual identification and selection of spermatozoa in a modified high-speed flow cytometer. For farm animal species, the technology is capable of producing sexed sperm at greater than 90% purity. However, only in the bovine, the technology has reached a developmental level that allows its commercial application. Meanwhile, the demand for female calves has grown rapidly, which encourages the demand for sex-sorted semen from high genetic value bulls. The success of the technology will depend mainly on the fertilizing capacity of the sorted spermatozoa, as this is the most affecting and economically relevant factor. To date, fertility is still variable and is quite dependent on post-sort processing. New processing techniques are under investigation and will likely be able to improve the fertility rates after AI with sex-sorted semen. It is of great importance to select the right bulls and to test the sorted samples on a routine basis. In addition to the demand for sex-sorted semen by the cattle industry, there is also a significant demand expressed by pig farmers. However, it is still unknown if the use of sex-sorted semen through commercial pig AI will be economically feasible. For the pig, the combination of in vitro fertilization with sexed semen and non-surgical embryo transfer is an alternative that merits further scientific attention. Recent developments in ovine AI and ET will make it very likely that commercial sheep industry will adopt the sexing technology in their breeding concepts. PMID:18638144

  8. Possible use of porphycenes as a membrane marker for flow cytometric detection of micronuclei

    SciTech Connect

    Wessels, J.M.; Nuesse, M. )

    1993-01-01

    For flow cytometric analysis of micronuclei induced by ionizing radiation or chemicals, debris plays an important role for the unambiguous detection of micronuclei. Several flow cytometric parameters have been used for a discrimination of micronuclei and debris (Schreiber et al., Cytometry 13:90-102, 1992). The lipophilic character of porphycenes can be used to selectively stain cellular and nuclear membranes. This chemical property of porphycenes requires the use of liposomes as a carrier. Porphycenes show high extinction coefficients at 360 nm and high fluorescence quantum yields between 600 nm and 750 nm. Due to their excellent photophysical properties they can therefore be used as a fluoroscent dye in combination with DNA specific dyes. Porphycenes (i.e. hydroxyethyl-tris(methoxyethyl)porphycene, HEPn) were used to either stain selectively the debris produced by membrane particles or to stain the membranes of nuclei and micronuclei for a better discrimination of debris and micronuclei.

  9. New optical configuration for flow cytometric sorting of aspherical cells

    NASA Astrophysics Data System (ADS)

    Sharpe, John C.; Schaare, Peter N.; Kuennemeyer, Rainer

    1997-05-01

    The orthogonal axes of illumination, flow, and detection in conventional sorting flow cytometers can limit accuracy or throughput when making fluorescence measurements on a spherical cells. A new radially symmetric optical configuration has been designed to overcome these problems. Both illumination and fluorescence collection are performed by a single optical element which encircles the sample stream flow axis. Unlike existing epi-illumination flow cytometer designs, these optics are compatible with electrostatic sorting. The resolution of this system is currently being evaluated for DNA chromosome content measurement with an ultimate goal of separation of X- and Y- chromosome-bearing mammalian spermatozoa. We describe the new optical configuration and present preliminary results of instrument performance. Comparison with a conventional orthogonal optical geometry is made using fluorescent microspheres, chicken red blood cells and chinchilla sperm.

  10. Fluoresceinated phosphoethanolamine for flow-cytometric measurement of lipid peroxidation.

    PubMed

    Maulik, G; Kassis, A I; Savvides, P; Makrigiorgos, G M

    1998-10-01

    A new lipophilic fluorescein probe (fluor-DHPE) has been identified that can assay lipid peroxidation in mammalian cells on a cell-by-cell or selected-cell-subpopulation basis by flow cytometry. Application of this approach requires that the fluorescent probe be nonexchangeable among cells. Fluorescein is an appropriate fluorophore, since its fluorescence matches the specifications of common flow cytometers and the compound loses its fluorescence upon reaction with peroxyl radicals. Upon examination of four lipophilic derivatives of fluorescein, fluor-DHPE was found to be the only probe that was nonexchangeable among labeled and unlabeled rat RBC for at least 24 h. The exposure of fluor-DHPE-labeled RBC to benzoyl peroxide followed by mixing the sample with RBC unexposed to peroxide led to a decrease in fluorescence. Furthermore, the flow cytometer could clearly select the subpopulation of cells undergoing lipid peroxidation from those cells that were not. Fluor-DHPE-labeled-RBC obtained from rats and exposed to cumene hydroperoxide also displayed a gradual decrease in fluorescence. This decrease was preventable by either regulation of the vitamin E content in the animal diet or in vitro supplementation of cells with vitamin E. We conclude that fluor-DHPE is a stable and nonexchangeable probe for monitoring lipid peroxidation in cell subpopulations by flow cytometry. PMID:9801063

  11. Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells.

    PubMed

    Singer, Benjamin D; Mock, Jason R; D'Alessio, Franco R; Aggarwal, Neil R; Mandke, Pooja; Johnston, Laura; Damarla, Mahendra

    2016-05-01

    Flow cytometry is a powerful tool capable of simultaneously analyzing multiple parameters on a cell-by-cell basis. Lung tissue preparation for flow cytometry requires creation of a single-cell suspension, which often employs enzymatic and mechanical dissociation techniques. These practices may damage cells and cause cell death that is unrelated to the experimental conditions under study. We tested methods of lung tissue dissociation and sought to minimize cell death in the epithelial, endothelial, and hematopoietic lineage cellular compartments. A protocol that involved flushing the pulmonary circulation and inflating the lung with Dispase, a bacillus-derived neutral metalloprotease, at the time of tissue harvest followed by mincing, digestion in a DNase and collagenase solution, and filtration before staining with fluorescent reagents concurrently maximized viable yields of epithelial, endothelial, and hematopoietic lineage cells compared with a standard method that did not use enzymes at the time of tissue harvest. Flow cytometry identified each population-epithelial (CD326(+)CD31(-)CD45(-)), endothelial (CD326(-)CD31(+)CD45(-)), and hematopoietic lineage (CD326(-)CD31(-)CD45(+))-and measured cellular viability by 7-aminoactinomycin D (7-AAD) staining. The Dispase method permitted discrimination of epithelial vs. endothelial cell death in a systemic lipopolysaccharide model of increased pulmonary vascular permeability. We conclude that application of a dissociative enzyme solution directly to the cellular compartments of interest at the time of tissue harvest maximized viable cellular yields of those compartments. Investigators could employ this dissociation method to simultaneously harvest epithelial, endothelial, and hematopoietic lineage and other lineage-negative cells for flow-cytometric analysis. PMID:26944088

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

    PubMed Central

    Cassart, Dominique; Fett, Thomas; Sarlet, Michaël; Baise, Etienne; Coignoul, Freddy; Desmecht, Daniel

    2007-01-01

    Background The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. Results The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. Conclusion Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction. PMID:17903245

  13. Flow cytometric fluorescence lifetime analysis of DNA binding fluorochromes

    SciTech Connect

    Crissman, Harry A.; Cui, H. H.; Steinkamp, J. A.

    2002-01-01

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

  14. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

    PubMed Central

    Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  15. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking.

    PubMed

    Whiten, D R; San Gil, R; McAlary, L; Yerbury, J J; Ecroyd, H; Wilson, M R

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  16. Intra- and interboar variability in flow cytometric sperm sex sorting.

    PubMed

    Alkmin, Diego V; Parrilla, Inmaculada; Tarantini, Tatiana; Parlapan, Laura; Del Olmo, David; Vazquez, Juan M; Martinez, Emilio A; Roca, Jordi

    2014-08-01

    To improve the efficiency of porcine sperm sex sorting using flow cytometry, the aims of the present study were to determine the relevance of inter- and intraboar variability in sperm sortability and to evaluate the significance of ejaculate semen characteristics in such variability. In addition, the variability among boars in the ability of sex-sorted spermatozoa to survive liquid storage at 15 °C to 17 °C was also evaluated. In total, 132 ejaculates collected from 67 boars of different breeds that were housed at an artificial insemination center were used in three experiments. X- and Y-chromosome-bearing sperm were simultaneously separated according to the Beltsville sperm-sorting technology using a high-speed flow cytometer. In the first experiment, interboar variability in the ability of the ejaculated spermatozoa to undergo the flow-based sex-sorting procedure was observed; the ejaculates of nearly 15% of the boars (n = 67) did not exhibit well-defined X- and Y-chromosome-bearing spermatozoa peaks in the histogram, and the ejaculate sperm concentration demonstrated good predictive value for explaining this variation, as indicated by the area under the receiver operating characteristics curve (0.88, P < 0.001). In the second experiment, a certain degree of intraboar variability was observed only in the boars that showed poor sperm sortability (measured according to the presence or not a well-defined split together with sperm sortability parameters) in the first ejaculate (n = 3). In contrast, boars classified as having good sperm sortability in the first ejaculate (n = 5) maintained this condition in five ejaculates collected over the subsequent 5 months. In the third experiment, sex-sorted spermatozoa from boars with good sperm sortability (n = 5) remained viable and motile (above 70% in all boars) after 48 hours of storage at 15 °C to 17 °C, which may facilitate the commercial application of sex-sorted spermatozoa in swine artificial insemination programs

  17. Flow cytometric measurement of pollutant stresses on algal cells

    SciTech Connect

    Berglund, D.L.; Eversman, S.

    1988-03-01

    The lichen Usnea fulvoreagens (Raes). Raes. was treated with four pH levels (5.5, 4.5, 3.5, and 2.5) of simulated acid rain (sulfuric acid, nitric acid, and a 1:1 combination of both) and automobile exhaust. The samples were dissociated and analyzed by a Becton-Dickinson FACS 440 flow cytometer. Analyses included measurement of chlorophyll autofluorescence and fluorescence due to uptake of fluorescein diacetate (FDA) and calcofluor white M2R (CFW). Cell parameters measured were esterase activity (FDA), membrane permeability (FDA, CFW), and intracellular pH (FDA). Mean fluorescence intensity from FDA staining and numbers of events were incorporated with autofluorescence information to produce a stress index of relative cell stress. Results indicated that highly stressed samples (lower pH treatments and greater exposure to exhaust) exhibited a low stress index of FDA fluorescence.Au

  18. Flow cytometric detection of Cryptosporidium oocysts in human stool samples.

    PubMed Central

    Valdez, L M; Dang, H; Okhuysen, P C; Chappell, C L

    1997-01-01

    Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection. PMID:9230372

  19. Flow cytometric measurement of total DNA and incorporated halodeoxyuridine

    DOEpatents

    Dolbeare, F.A.; Gray, J.W.

    1983-10-18

    A method for the simultaneous flow cylometric measurement of total cellular DNA content and of the uptake of DNA precursors as a measure of DNA synthesis during various phases of the cell cycle in normal and malignant cells in vitro and in vivo is described. The method comprises reacting cells with labelled halodeoxyuridine (HdU), partially denaturing cellular DNA, adding to the reaction medium monoclonal antibodies (mabs) reactive with HdU, reacting the bound mabs with a second labelled antibody, incubating the mixture with a DNA stain, and measuring simultaneously the intensity of the DNA stain as a measure of the total cellular DNA and the HdU incorporated as a measure of DNA synthesis. (ACR)

  20. High Content Flow Cytometric Micronucleus Scoring Method is Applicable to Attachment Cell Lines

    PubMed Central

    Bryce, Steven M.; Shi, Jing; Nicolette, John; Diehl, Marilyn; Sonders, Paul; Avlasevich, Svetlana; Raja, Sarojini; Bemis, Jeffrey C.; Dertinger, Stephen D.

    2009-01-01

    A flow cytometric method for analyzing suspension cell cultures for micronucleus content has been previously reported [Environ. Molec. Mutagen. 47 (2006) 56–66]. The experiments described herein were undertaken to evaluate the compatibility of this method (In Vitro MicroFlow®) with attachment cells. Initially, CHO-K1 cells were studied in nine independent experiments using mitomycin C and cyclophosphamide. The results demonstrated the effectiveness of the cell processing procedure, and also provided historical control data that were useful for setting criteria for making positive calls. Subsequently, CHO-K1 cells were treated with methyl methanesulfonate, mitomycin C, etoposide, vinblastine sulfate, dexamethasone, and sodium chloride. Whereas the four genotoxicants were each observed to increase micronucleus frequencies, the non-genotoxicants induced no such response up to cytotoxic concentrations. Following this initial work, inter-laboratory transferability was evaluated across three sites using a common cell staining and analysis protocol for CHO-K1 or V79 cells that had been treated with the ten chemicals listed in Annex 3 of the OECD Draft Proposal for a New Guideline 487: In Vitro Mammalian Cell Micronucleus Test. With the exception of benzo[a]pyrene at one site, each laboratory observed increased micronucleus frequencies for the genotoxicants, whereas no significant induction occurred with the non-genotoxicants. Interestingly, the method appeared to distinguish between genotoxic modes of action, as only aneugens increased the average micronucleus fluorescence intensity and the frequency of hypodiploid nuclei. Collectively, these data suggest that flow cytometry is capable of providing reliable micronucleus counts, and that additional information is obtained that appears to discern genotoxic modes of action. PMID:19950402

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

    SciTech Connect

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

    1986-12-01

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

  2. Collection, isolation, and flow cytometric analysis of human endocervical samples.

    PubMed

    Juno, Jennifer A; Boily-Larouche, Genevieve; Lajoie, Julie; Fowke, Keith R

    2014-01-01

    Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina. PMID:25045942

  3. Amplified flow-cytometric separation-free fluorescence immunoassays

    SciTech Connect

    Saunders, G.C.; Jett, J.H.; Martin, J.C.

    1985-12-01

    An equilibrium-type competitive-binding fluorescence immunoassay with high sensitivity and excellent precision is described that obviates separation of free from bound label. In the assay relatively large (10 microns diameter) antibody-coated non-fluorescent particles and very small (0.10 micron diameter) antigen-coated fluorescent latex particles are used. Soluble nonlabeled antigen competes with antigen on the microspheres for antibody binding sites on the larger spheres. After equilibrium is attained, the fluorescence distribution of 5000 of the large spheres is measured in a flow cytometer. The mean values for the fluorescence distribution obtained from samples containing known concentrations of soluble antigen are used to construct a standard displacement curve. In a prototype assay for the antigen horseradish peroxidase, a sensitivity of 10(-12) mol/L has been achieved. Undiluted serum can be assayed without loss of sensitivity. Preliminary experiments also indicate that double-antibody sandwich-type assays of very high sensitivity (10(-14) mol/L) are also possible when this dual-sphere concept is exploited.

  4. A flow cytometric approach to assess phytoplankton respiration.

    PubMed

    Grégori, Gérald; Denis, Michel; Lefèvre, Dominique; Beker, Beatriz

    2002-01-01

    Microbial respiration in the ocean is considered as the major process representative of the organic matter biological oxidation. The corresponding metabolic CO2 production was estimated to be about 22 Pg C y(-1). However, the in situ respiration rate is generally too low (by several orders of magnitude) to be accessible to the available direct measurement methods. Some fluorescent probes, such as DiOC6(3) (Molecular Probes, USA) have been shown to be very sensitive to changes in the proton electrochemical potential difference (DeltamuH+), characterising mitochondrial and plasmic membranes bearing the cell respiratory system in eukaryotic and prokaryotic cells respectively. In mitochondria, DeltamuH+ is linked to the flux of oxygen uptake by a linear relationship. To our knowledge, no such relationship has been established in the case of whole marine cells. In the present work, we addressed the dark respiration rate of the Chlorophyceae Dunaliella tertiolecta (Butcher) in axenic cultures, both directly by using a highly sensitive oxygraph (Oroboros) and by staining cells with DiOC6(3). We found and standardized a linear relationship between oxygen uptake by D. tertiolecta and its green fluorescence induced by DiOC6(3), enabling the determination by flow cytometry of the respiration rate of D. tertiolecta. PMID:12815298

  5. Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

    PubMed Central

    Juno, Jennifer A.; Boily-Larouche, Genevieve; Lajoie, Julie; Fowke, Keith R.

    2014-01-01

    Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina. PMID:25045942

  6. A rapid, simple and sensitive flow cytometric system for detection of Plasmodium falciparum.

    PubMed

    Saito-Ito, A; Akai, Y; He, S; Kimura, M; Kawabata, M

    2001-11-01

    We have established a rapid, simple and sensitive flow cytometric system for the detection of Plasmodium falciparum that involves lysing erythrocytes and staining parasites at the same time using a newly developed hemolysing and staining solution containing dodecyl methyl ammonium chloride and acridine orange. In this system, freed parasites of P. falciparum could be plotted separately from erythrocyte ghosts, white blood cells and platelets on the two-dimensional scattergram of forward-angle light scatter and green fluorescence by flow cytometry with an argon laser. It took only 2-3 min per sample to obtain the scattergram and analyze the data, including the time of sample preparation for flow cytometric analysis. Sample preparation with this method does not require any difficult handling procedures. The threshold of parasite detection was almost equal to that of microscopic examination for cultured P. falciparum. The results of drug-susceptibility assays using this system were also almost identical to those obtained using microscopic examination. In this system, parasites at different erythrocytic stages could be easily distinguished. This system must prove useful and practical for basic laboratory studies of P. falciparum including those requiring the differential measurement of parasites at specific erythrocytic stages. PMID:11719111

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

    NASA Technical Reports Server (NTRS)

    Li, Z. K.

    1985-01-01

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

  8. Flow cytometric characterization of rat thymus cells in a radiation-dominated model of combined injury

    SciTech Connect

    Kaffenberger, W.; Gruber, D.F.; MacVittie, T.J.

    1988-05-01

    Thymuses of rats that had been: a) gamma-irradiated (500 cGy whole-body radiation (R)), or b) thermally injured (20% BSA dorsal, scald burn (TI)), or c) combined injured (irradiation followed by burn (CI)) were studied for involution and recovery processes after sublethal treatments. The expression of surface antigens on thymic cells before and after injuries was evaluated using the monoclonal antibodies (mcAB) MRC OX4, MRC OX7, MRC OX8, W3/13 HLK, and W3/25 and flow cytometric analysis. Thymic cellularity decreased to less than 1% of normal (N), age-matched rats by 4 days after R or CI. Recovery reached 60% to 70% of N by 28 days post treatments. TI caused a biphasic thymic recovery pattern with nadirs of 40% of N on days 7 and 21. Recovery at day 28 was similar to that after R and CI. Expression of OX7, OX8, W3/13, and W3/25 antigens all reached nadirs of 40% of N by day 4 after R and CI. Recovery of antigen expression, except for W3/25, was near completion by day 7 after R and CI. Changes in antigen expression after TI were less pronounced for all mcAB tested. Decreases in labeling of thymocytes with the helper T-cell marker, W3/25, observed after TI, could not be correlated with elevated expressions of the suppressor/cytotoxic T-lymphocyte antigen, OX8. Variations in relative labeling of nonlymphoid thymic cells with OX4 (Ia-antigen) reflected the disappearance and recovery of radiosensitive lymphoid thymocytes. The similarity of results after R and CI demonstrate that the model of CI is radiation-dominated. The addition of burn injury to radiation trauma had no synergistically damaging effect on the parameters studied.

  9. Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future.

    PubMed

    Avlasevich, Svetlana; Bryce, Steven; De Boeck, Marlies; Elhajouji, Azeddine; Van Goethem, Freddy; Lynch, Anthony; Nicolette, John; Shi, Jing; Dertinger, Stephen

    2011-01-01

    The relative simplicity of the in vitro micronucleus (MNvit) endpoint has made it amenable to several automated scoring approaches. Flow cytometry is one such scoring platform that has been successfully employed. This review describes the origins of the MNvit assay, as well as the evolution and properties of flow cytometry-based scoring systems. While the current state-of-the-art methods acquire micronucleus (MN) frequency data very efficiently, it is becoming clear that they also endow the assay with high information content. For instance, simultaneous with MN frequency determinations, several additional endpoints are acquired that provide insights into cytotoxicity, cell cycle perturbations and, in the event of MN induction, information about genotoxic mode of action. This review concludes with a discussion regarding data gaps and also recommendations for additional work that is needed to more fully realise the potential of flow cytometric MNvit scoring. PMID:21164196

  10. Flow cytometric determination of leukemia-associated marker combinations for the study of minimal residual disease.

    PubMed

    Babusíková, O; Glasová, M; Kusenda, J; Koníková, E; Mésárosová, A

    1994-01-01

    To study the minimal residual disease in acute leukemia patients we used some marker combinations related either to the simultaneous surface membrane and cytoplasmic marker expression, or to the expression of atypical marker combinations, that are absent or extremely rare in normal hematopoietic cells. We investigated to which extent flow cytometric analysis of leukemia-associated marker combination may contribute to sensitive follow-up in patients with acute leukemia. For this purpose dilution experiments were performed, in which artificial mixtures of normal peripheral blood lymphocytes and leukemia cells from a patient with leukemia-associated phenotype were prepared and analyzed for double positive cells. Our results showed that the sensitivity of double color immunofluorescence assay was 3 in 10(4) cells. Sequential studies of residual disease were evaluated in five acute leukemia patients with leukemia-associated markers combinations at diagnosis. In three of them morphologic relapse was preceded by the immunologic detection of small amounts of leukemia cells, while in two other cases, in which no double positive cells for leukemia-associated markers were found, patients are still in hematologic remission. This approach to the study of minimal residual disease could be valuable in monitoring the efficiency of chemotherapy, as well as in evaluating the quality control of bone marrow before autografting. Furthermore, flow cytometric approach can efficiently complete other methods, which are used for more exact definition of remission in acute leukemia patients. PMID:7870213

  11. Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

    SciTech Connect

    Elstein, K.H.; Thomas, D.J.; Zucker, R.M.

    1995-10-01

    Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we observed a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 {mu}M tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 {mu}M dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. 37 refs., 3 figs., 1 tab.

  12. Flow cytometric characterization of microglia in the offspring of PolyI:C treated mice.

    PubMed

    Manitz, Marie Pierre; Plümper, Jennifer; Demir, Seray; Ahrens, Maike; Eßlinger, Manuela; Wachholz, Simone; Eisenacher, Martin; Juckel, Georg; Friebe, Astrid

    2016-04-01

    The neuropathology of schizophrenia has been reported to be closely associated with microglial activation. In a previous study, using the prenatal PolyI:C schizophrenia animal model, we showed an increase in cell numbers and a reduction in microglial branching in 30-day-old PolyI:C descendants, which suggests that there is microglial activation during adolescence. To provide more information about the activation state, we aimed to examine the expression levels of Iba1, which was reported to be up-regulated in activated microglia. We used a flow cytometric approach and investigated CD11b and CD45, two additional markers for the identification of microglial cells. We demonstrated that intracellular staining against Iba1 can be used as a reliable flow cytometric method for identification of microglial cells. Prenatal PolyI:C treatment had long-term effects on CD11b and CD45 expression. It also resulted in a trend towards increased Iba1 expression. Imbalance in CD11b, CD45, and Iba1 expression might contribute to impaired synaptic surveillance and enhanced activation/inflammatory activity of microglia in adult offspring. PMID:26872595

  13. Flow Cytometric Method for the Detection of Flavonoids in Cell Lines.

    PubMed

    Grootaert, Charlotte; Gonzales, Gerard Bryan; Vissenaekens, Hanne; Van de Wiele, Tom; Raes, Katleen; Smagghe, Guy; Van Camp, John

    2016-09-01

    Here, we describe an easy-to-use flow cytometric method using diphenylboric acid 2-amino ethyl ester (DPBA) stain for the detection of flavonoids in cells from human/animal origin. Flavonoid bioavailability and bioactivity depend on structure, conjugation and the cell type to which they are presented. We have studied cellular uptake of five flavonoids with different structures and conjugation forms. First, parameters including fixation method, technical and batch variability, and concentration were optimized. Second, uptake of two aglycones-quercetin and hesperetin-and their corresponding glycosides-rutin and hesperidin-in Caco-2 cells was compared. Third, the aglycone quercetin, glycoside rutin, and glucuronide baicalin were added to the Caco-2, HepG2, and CHO-K1 cell lines at 1, 10, and 20 µM concentrations and cellular uptake was measured after 1, 4, and 7 h. We conclude that quercetin was taken up by cells in a dose-dependent way, and that HepG2 cells had the highest uptake factors, followed by CHO-K1 and Caco-2 cells. Confocal microscopy showed cell type-dependent localization of quercetin in the cell membrane and cytoplasm. No uptake of flavonoid glycosides was detected. This flow cytometric method can be used for future research unravelling mechanisms behind flavonoid bioactivity in health and disease at the cellular level. PMID:27280551

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

    SciTech Connect

    Feldhaus, Michael ); Siegel, Robert W. ); Opresko, Lee ); Coleman, James R. ); Feldhaus, Jane M. ); Yeung, Yik A.; Cochran, Jennifer R.; Heinzelman, Peter; Colby, David; Swers, Jeffrey; Graff, Christilyn; Wiley, H Steven ); Wittrup, K D.

    2003-02-28

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

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

    PubMed Central

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

    2015-01-01

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

  16. Genotoxic mode of action predictions from a multiplexed flow cytometric assay and a machine learning approach.

    PubMed

    Bryce, Steven M; Bernacki, Derek T; Bemis, Jeffrey C; Dertinger, Stephen D

    2016-04-01

    Several endpoints associated with cellular responses to DNA damage as well as overt cytotoxicity were multiplexed into a miniaturized, "add and read" type flow cytometric assay. Reagents included a detergent to liberate nuclei, RNase and propidium iodide to serve as a pan-DNA dye, fluorescent antibodies against γH2AX, phospho-histone H3, and p53, and fluorescent microspheres for absolute nuclei counts. The assay was applied to TK6 cells and 67 diverse reference chemicals that served as a training set. Exposure was for 24 hrs in 96-well plates, and unless precipitation or foreknowledge about cytotoxicity suggested otherwise, the highest concentration was 1 mM. At 4- and 24-hrs aliquots were removed and added to microtiter plates containing the reagent mix. Following a brief incubation period robotic sampling facilitated walk-away data acquisition. Univariate analyses identified biomarkers and time points that were valuable for classifying agents into one of three groups: clastogenic, aneugenic, or non-genotoxic. These mode of action predictions were optimized with a forward-stepping process that considered Wald test p-values, receiver operator characteristic curves, and pseudo R(2) values, among others. A particularly high performing multinomial logistic regression model was comprised of four factors: 4 hr γH2AX and phospho-histone H3 values, and 24 hr p53 and polyploidy values. For the training set chemicals, the four-factor model resulted in 94% concordance with our a priori classifications. Cross validation occurred via a leave-one-out approach, and in this case 91% concordance was observed. A test set of 17 chemicals that were not used to construct the model were evaluated, some of which utilized a short-term treatment in the presence of a metabolic activation system, and in 16 cases mode of action was correctly predicted. These initial results are encouraging as they suggest a machine learning strategy can be used to rapidly and reliably predict new chemicals

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

    USGS Publications Warehouse

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

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  20. A Flow Cytometric Method for Rapid Selection of Novel Industrial Yeast Hybrids

    PubMed Central

    Bell, P. J. L.; Deere, D.; Shen, J.; Chapman, B.; Bissinger, P. H.; Attfield, P. V.; Veal, D. A.

    1998-01-01

    We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 × 106 cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains. PMID:9572934

  1. Flow cytometric detection of altered signaling in myelodysplastic syndrome and cytopenia.

    PubMed

    Gao, Juehua; Swaminathan, Suchitra; Pai, Navin; Johnson, Zachary; Chen, Yi-Hua; Peterson, LoAnn; Goolsby, Charles

    2015-12-01

    Multiparameter flow cytometric analysis allows for precise evaluation of growth factor stimulated intracellular signaling in distinct immunophenotype defined hematopoetic populations. Our analysis of intracellular phosphoprotein in response to major hematopoietic growth factors or cytokines showed several interesting findings. Although there was no characteristic signaling abnormality that was diagnostic for MDS, MDS cases were often associated with more signaling aberrancies involving more cellular populations. Higher than average response in the CD34(+)CD117(+) progenitor cells to Flt3 ligand and stem cell factor stimulation was frequently associated with high risk features or disease progression in MDS. Although preliminary results hint an adverse prognostic role of dysregulated FLT3 pathway in MDS cases, whether this observation adds independent prognostic value to the existing prognostic system needs to be further explored in future prospective studies. PMID:26410459

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

    NASA Astrophysics Data System (ADS)

    Melzer, Susanne; Bocsi, Jozsef; Tárnok, Attila

    2015-03-01

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

  3. Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

    PubMed Central

    Baker, Gregory J.; Castro, Maria G.; Lowenstein, Pedro R.

    2016-01-01

    Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the β-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4–6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists’ discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the

  4. Development of a Robust Flow Cytometric Assay for Determining Numbers of Viable Bacteria

    PubMed Central

    Jepras, R. I.; Carter, J.; Pearson, S. C.; Paul, F. E.; Wilkinson, M. J.

    1995-01-01

    Several fluorescent probes were evaluated as indicators of bacterial viability by flow cytometry. The probes monitor a number of biological factors that are altered during loss of viability. The factors include alterations in membrane permeability, monitored by using fluorogenic substrates and fluorescent intercalating dyes such as propidium iodide, and changes in membrane potential, monitored by using fluorescent cationic and anionic potential-sensitive probes. Of the fluorescent reagents examined, the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC(inf4)(3)] proved the best candidate for use as a general robust viability marker and is a promising choice for use in high-throughput assays. With this probe, live and dead cells within a population can be identified and counted 10 min after sampling. There was a close correlation between viable counts determined by flow cytometry and by standard CFU assays for samples of untreated cells. The results indicate that flow cytometry is a sensitive analytical technique that can rapidly monitor physiological changes of individual microorganisms as a result of external perturbations. The membrane potential probe DiBAC(inf4)(3) provided a robust flow cytometric indicator for bacterial cell viability. PMID:16535078

  5. Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study.

    PubMed

    Kasamoto, Sawako; Mukai, Daisuke; Masumori, Shoji; Suzuki, Kenichiro; Tanaka, Ryota; Torous, Dorothea K; Yamate, Jyoji; Hayashi, Makoto

    2014-03-01

    In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague-Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlow(PLUS) Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r(2)=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r(2)=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power. PMID:24548793

  6. A flow cytometric approach to the study of crustacean cellular immunity

    USGS Publications Warehouse

    Cardenas, W.; Jenkins, J.A.; Dankert, J.R.

    2000-01-01

    Responses of hemocytes from the crayfish Procambarus zonangulus to stimulation by fungal cell walls (Zymosan A) were measured by flow cytometry. Changes in hemocyte physical characteristics were assessed flow cytometrically using forward- and sidescatter light parameters, and viability was measured by two-color fluorescent staining with calcein-AM and ethidium homodimer 1. The main effects of zymosan A on crayfish hemocytes were reduction in cell size and viability compared to control mixtures (hemocytes in buffer only). Adding diethyldithiocarbamic acid, an inhibitor of phenoloxidase, to hemocyte to zymosan mixtures delayed the time course of cell size reduction and cell death compared to zymosan-positive controls. The inclusion of trypsin inhibitor in reaction mixtures further delayed the reduction in hemocyte size and cell death, thereby indicating that a proteolytic cascade, along with prophenoloxidase activation, played a key role in generating signal molecules which mediate these cellular responses. In addition to traditional methods such as microscopy and protein chemistry, flow cytometry can provide a simple, reproducible, and sensitve method for evaluating invertebrate hemocyte responses to immunological stimuli.

  7. Flow cytometric evaluation of disseminated intravascular coagulation in a canine endotoxemia model

    PubMed Central

    Yu, Dohyeon; Noh, Dongho; Park, Jinho

    2015-01-01

    Sepsis is associated with substantial morbidity and mortality in dogs. Alterations in hemostasis by systemic inflammation play an important role in the pathophysiology of sepsis. To evaluate the functional hemostatic changes in sepsis, we evaluated coagulation profiles and flow cytometric measurement of P-selectin (CD62P) expression on platelets, as well as platelet-leukocyte aggregation from a lipopolysaccharide (LPS)-induced endotoxemia model in dogs (n = 7). A sublethal dose of LPS [1 mg/kg body weight (BW)] induced thrombocytopenia and increased activated partial thromboplastin time (aPTT), prothrombin time (PT), and D-dimer concentrations. Flow cytometry analysis showed a significant increase in P-selectin expression on platelets between 1 and 24 h of a total 48 h of the experiment. In addition, platelet-leukocyte aggregation was significantly increased in the early stage of endotoxemia (at 1 and < 6 h for platelet-monocyte aggregation and at 3 h for platelet-neutrophil aggregation). Our results suggest that CD62P expression on platelets and platelet-leukocyte aggregation, as measured by flow cytometry, can be useful biomarkers of disseminated intravascular coagulation (DIC) in canine sepsis. These functional changes contribute to our understanding of the pathophysiology of hemostasis in endotoxemia. PMID:25673909

  8. Relationship of flow cytometric sperm integrity assessments with boar fertility performance under optimized field conditions.

    PubMed

    Broekhuijse, M L W J; Šoštarić, E; Feitsma, H; Gadella, B M

    2012-12-01

    The number of intact and functional spermatozoa in semen can be assessed with flow cytometry and is believed to relate to male fertility. The aim of this study was to examine whether currently used sperm integrity assessments with flow cytometry correlate with field fertility data obtained for boar semen. For this purpose, 20 boars were followed for a 20-wk period (with a total average production of 33 ejaculates per boar) and the obtained fertility results (farrowing rate and number of piglets born) of commercial artificial insemination doses made from these ejaculates were recorded. Fertility results were corrected for farm, sow, boar, and semen-related parameters. From the same semen samples, sperm cell integrity was assessed with respect to DNA and to membrane integrity, acrosome intactness and responsiveness, and mitochondrial potential using established flow cytometric assays. This was done on freshly produced semen and on semen stored for up to 15 d. Remarkably, none of the individual membrane integrity variables was significantly related to fertility results. In contrast, the amount of DNA damage as assessed at 7 to 10 d and at 14 to 15 d of semen storage related to farrowing rate (P = 0.0400) and total number of piglets born (P = 0.0310), respectively. Therefore, the degree of DNA damage in stored boar semen samples may be a useful factor to evaluate semen as an indicator for litter size and farrowing rate. PMID:23255815

  9. Expression of oestrogen and progesterone receptors in gastric cancer: a flow cytometric study.

    PubMed

    Karat, D; Brotherick, I; Shenton, B K; Scott, D; Raimes, S A; Griffin, S M

    1999-06-01

    Increased expression of oestrogen (ER) and progesterone (PR) receptors have been reported in gastric adenocarcinoma, although results have been variable. Immunohistochemical staining methodologies, in particular in the detection of ER, have been inconsistent with many tumours being classified ER-negative. In this study we have used flow cytometry to quantify expression of ER and PR in gastric adenocarcinoma and examine their relationships with established prognostic indicators. Cytokeratin-positive cells obtained from tumour biopsies of 50 patients with gastric cancer and ten control patients were labelled with biotinylated ER or PR antibodies followed by streptavidin PE. Flow cytometry was seen to increase the detection of ER levels in gastric cancer with more receptor-positive patients in this study than in results published to date. We believe this is related to the sensitivity of the flow cytometric assay with the detection of small shifts in ER level detected using cytokeratin gating. On analysis, the data showed no significant correlations with tumour stage and grade, and no differences were seen between normal mucosa and gastric cancer samples. PMID:10376983

  10. Flow cytometric determination of bacterial populations in bottled natural mineral waters

    NASA Astrophysics Data System (ADS)

    Beisker, Wolfgang; Meier, H.

    1998-04-01

    In order to enhance the quality and safety of bottled natural mineral waters, new methodologies besides classical bacteriology have been evaluated. Multi laser flow cytometry has been used to identify bacterial populations based on their DNA content, physiological activity and phylogeny from in situ hybridization with rRNA targeted DNA probes. Due to the low content of organic material in these waters, the bacterial population are under conditions (low ribosome content, low activity, etc.) which makes it hard to detect them flow cytometrically. The numbers of bacteria are in the range between 1000 and 100,000 per ml (for uncarbonated waters). Filtration techniques to enrich the bacterial population have been developed in combination with specific staining and hybridization protocols. First results on some selected brands show, that most bacteria belong to the beta subclass of proteobacteria. If the DNA containing cells (DAPI staining) are counted as 100%, 84% could be stained with a eubacteria probe. From these 84% 68% belong to the beta subclass, 8.2% to the alpha and 0.3% to the gamma subclass of roteobacteria. 8.5% could be identified as cytophaga flexibacter. By optimizing DNA staining with cyanine dyes and enhancing the sensitivity of light scatter detection, the detection limit could be considerably lowered.

  11. CUBIC: a three-dimensional colored projection of Consort 30 generated trivariate flow cytometric data.

    PubMed

    Greimers, R; Rongy, A M; Schaaf-Lafontaine, N; Boniver, J

    1991-01-01

    The CUBIC program displays three-dimensional colored dot plots of flow cytometric trivariate data collected by unmodified commercial instruments (FACScan flow cytometer, FACS 440 cell sorter). Assuming a bimodal distribution of the fluorescence intensity of the cells, the eight theoretical subpopulations involved in a three-color fluorescence histogram are clearly localized in the 3-D space by colored dots that are clustered near each corner of a cubic frame. Rotation, tilting, and zoom functions are available. Table look-up is not needed. CUBIC was illustrated by two experiments: 1) three-color immunofluorescence of antigens on human lymphocytes using monoclonal antibodies conjugated either to fluorescein (FITC), to R-phycoerythrin (PE), or to biotin revealed by a streptavidin coupled to a PE-Texas red tandem conjugate (TC); 2) two-color immunofluorescence of CD4 and CD8 antigens on thymocytes of healthy or preleukemic mice correlated to the DNA content quantified by 7-amino-actinomycin D (7-AAD). The three fluorescences were excited by a single argon-ion laser emitting at 488 nm. PMID:1764980

  12. Differentiation of A31T6 proadipocytes to adipocytes: A flow cytometric analysis

    SciTech Connect

    Smyth, M.J.; Wharton, W. )

    1992-03-01

    A flow cytometric assay has been developed which provides precise, quantitative information on the accumulation of cytoplasmic triglycerides in individual A31T6 proadipocytes as they differentiate into adipocytes. The opportunity to measure multiple optical parameter on a cell-by-cell basis has enabled us to monitor phenotypic aspects of differentiation with a greater level of sensitivity than was previously possible. Using the fluorescent hydrophobic probe, Nile red, they have found that as a cell proceeds along the differentiation pathway, the gold fluorescence signal from the cell increases, reflecting the accumulation of cytoplasmic lipid droplets. They have determined (1) the presence of an undifferentiated population of cells whose existence is not detected by conventional phase microscopy, (2) that insulin is no required to drive differentiation in this system, (3) that exposure to a combination of insulin and dexamethasone results in a lower accumulation of lipid in a cell than does exposure to either agent alone, and (4) that A31T6 cells show the same response to differentiation-promoting agents whether applied at the time of plating or at confluence.

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

    PubMed

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

    2015-10-01

    The majority of vaccines and several treatments are administered by intramuscular injection. The aim is to engage and activate immune cells, although they are rare in normal skeletal muscle. The phenotype and function of resident as well as infiltrating immune cells in the muscle after injection are largely unknown. While methods for obtaining and characterizing murine muscle cell suspensions have been reported, protocols for nonhuman primates (NHPs) have not been well defined. NHPs comprise important in vivo models for studies of immune cell function due to their high degree of resemblance with humans. In this study, we developed and systematically compared methods to collect vaccine-injected muscle tissue to be processed into single cell suspensions for flow cytometric characterization of immune cells. We found that muscle tissue processed by mechanical disruption alone resulted in significantly lower immune cell yields compared to enzymatic digestion using Liberase. Dendritic cell subsets, monocytes, macrophages, neutrophils, B cells, T cells and NK cells were readily detected in the muscle by the classic human markers. The methods for obtaining skeletal muscle cell suspension established here offer opportunities to increase the understanding of immune responses in the muscle, and provide a basis for defining immediate post-injection vaccine responses in primates. PMID:26099800

  14. Cytokinetic investigations in human breast cancer by flow cytometrically recorded DNA/protein distributions.

    PubMed

    Weiss, H; Görlich, M; Frege, J; Granetzny, A; Streller, B; Nitschke, U; Weiher, U

    1996-01-01

    This prospective study characterizes T1-T2 breast carcinomas (N = 114) and fibroadenomas (N = 16) by cell kinetic parameters derived from flow cytometrically recorded DNA/protein histograms. Ploidy level, cell cycle distribution and the number of cell subpopulations (SP) characterized by correlating DNA and protein values were assessed. The subpopulations were derived from the three-dimensional plot. The estrogen receptor (ER) status was determined biochemically (N = 61). Within the G1/0 cell peak 1-6 SP were evident in principle. Depending on the number of SP, two subsets were established: subset 1 with < or = 2 SP, subset 2 with > or = 3 SP. They differed significantly in proliferative activity expressed in the percentage of cells in the G2M phase. Subset 2 showed the higher activity. Analysis of subset distributions revealed that subset 1 prevails in favourable prognostic cases as ER positive cases (P < 0.03), lobular carcinomas (P < 0.01) and LN- cases (P < 0.03), whereas subset 2 prevails in the unfavourable counterparts. Analysis of variance showed that the main effect on proliferative activity indicated by G2M% is due to subpopulation composition rather than histologic type, nodal status or ER status (P < 0.01, P < 0.002, P < 0.05), not even due to ploidy level (P < 0.0001). The rationale for subset stratification may be cytogenetic variability connected with protein content heterogeneity accounting for kinetic SP. PMID:8789270

  15. Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment

    PubMed Central

    Lizotte, Patrick H.; Jones, Robert E.; Keogh, Lauren; Ivanova, Elena; Liu, Hongye; Awad, Mark M.; Hammerman, Peter S.; Gill, Ritu R.; Richards, William G.; Barbie, David A.; Bass, Adam J.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark; Wong, Kwok-Kin

    2016-01-01

    With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials. PMID:27539742

  16. Fine needle aspirate flow cytometric phenotyping characterizes immunosuppressive nature of the mesothelioma microenvironment.

    PubMed

    Lizotte, Patrick H; Jones, Robert E; Keogh, Lauren; Ivanova, Elena; Liu, Hongye; Awad, Mark M; Hammerman, Peter S; Gill, Ritu R; Richards, William G; Barbie, David A; Bass, Adam J; Bueno, Raphael; English, Jessie M; Bittinger, Mark; Wong, Kwok-Kin

    2016-01-01

    With the emergence of checkpoint blockade and other immunotherapeutic drugs, and the growing adoption of smaller, more flexible adaptive clinical trial designs, there is an unmet need to develop diagnostics that can rapidly immunophenotype patient tumors. The ability to longitudinally profile the tumor immune infiltrate in response to immunotherapy also presents a window of opportunity to illuminate mechanisms of resistance. We have developed a fine needle aspirate biopsy (FNA) platform to perform immune profiling on thoracic malignancies. Matching peripheral blood, bulk resected tumor, and FNA were analyzed from 13 mesothelioma patients. FNA samples yielded greater numbers of viable cells when compared to core needle biopsies. Cell numbers were adequate to perform flow cytometric analyses on T cell lineage, T cell activation and inhibitory receptor expression, and myeloid immunosuppressive checkpoint markers. FNA samples were representative of the tumor as a whole as assessed by head-to-head comparison to single cell suspensions of dissociated whole tumor. Parallel analysis of matched patient blood enabled us to establish quality assurance criteria to determine the accuracy of FNA procedures to sample tumor tissue. FNA biopsies provide a diagnostic to rapidly phenotype the tumor immune microenvironment that may be of great relevance to clinical trials. PMID:27539742

  17. Combined flow cytometric analysis of surface and intracellular antigens reveals surface molecule markers of human neuropoiesis.

    PubMed

    Turaç, Gizem; Hindley, Christopher J; Thomas, Ria; Davis, Jason A; Deleidi, Michela; Gasser, Thomas; Karaöz, Erdal; Pruszak, Jan

    2013-01-01

    Surface molecule profiles undergo dynamic changes in physiology and pathology, serve as markers of cellular state and phenotype and can be exploited for cell selection strategies and diagnostics. The isolation of well-defined cell subsets is needed for in vivo and in vitro applications in stem cell biology. In this technical report, we present an approach for defining a subset of interest in a mixed cell population by flow cytometric detection of intracellular antigens. We have developed a fully validated protocol that enables the co-detection of cluster of differentiation (CD) surface antigens on fixed, permeabilized neural cell populations defined by intracellular staining. Determining the degree of co-expression of surface marker candidates with intracellular target population markers (nestin, MAP2, doublecortin, TUJ1) on neuroblastoma cell lines (SH-SY5Y, BE(2)-M17) yielded a combinatorial CD49f(-)/CD200(high) surface marker panel. Its application in fluorescence-activated cell sorting (FACS) generated enriched neuronal cultures from differentiated cell suspensions derived from human induced pluripotent stem cells. Our data underlines the feasibility of using the described co-labeling protocol and co-expression analysis for quantitative assays in mammalian neurobiology and for screening approaches to identify much needed surface markers in stem cell biology. PMID:23826393

  18. A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

    PubMed

    Tennenbaum, T; Giloh, H; Fusenig, N E; Kapitulnik, J

    1988-06-01

    A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types. PMID:2453587

  19. LY303366 exhibits rapid and potent fungicidal activity in flow cytometric assays of yeast viability.

    PubMed

    Green, L J; Marder, P; Mann, L L; Chio, L C; Current, W L

    1999-04-01

    LY303366 is a semisynthetic analog of the antifungal lipopeptide echinocandin B that inhibits (1,3)-beta-D-glucan synthase and exhibits efficacy in animal models of human fungal infections. In this study, we utilized flow cytometric analysis of propidium iodide uptake, single-cell sorting, and standard microbiological plating methods to study the antifungal effect of LY303366 on Saccharomyces cerevisiae and Candida albicans. Our data indicate that an initial 5-min pulse treatment with LY303366 caused yeasts to take up propidium iodide and lose their ability to grow. Amphotericin B and cilofungin required longer exposure periods (30 and 180 min, respectively) and higher concentrations to elicit these fungicidal effects. These two measurements of fungicidal activity by LY303366 were highly correlated (r > 0.99) in concentration response and time course experiments. As further validation, LY303366-treated yeasts that stained with propidium iodide were unable to grow in single-cell-sorted cultures. Our data indicate that LY303366 is potent and rapidly fungicidal for actively growing yeasts. The potency and rapid action of this new fungicidal compound suggest that LY303366 may be useful for antifungal therapy. PMID:10103187

  20. A novel flow cytometric-based method to measure kinase inhibition in sputum from COPD subjects

    PubMed Central

    Nicholson, G C; Holloway, R A; Leaker, B R; Kilty, I; Salganik, M; Tan, L; Barnes, P J; Donnelly, L E

    2016-01-01

    Introduction Janus kinases (JAKs) regulate inflammatory gene expression through phosphorylation of signal transducer and activator of transcription (STAT) proteins. Expression of STAT proteins is increased in chronic obstructive pulmonary disease (COPD), and may be involved in driving chronic inflammation. Oral JAK inhibitors are effective as anti-inflammatory therapy but exhibit dose-limiting adverse effects. Development of inhaled compounds would be enhanced by robust biomarkers that directly reflect the anti-inflammatory and pharmacological activity in the lung. Methods A novel flow cytometry assay was developed to measure STAT1 phosphorylation in sputum inflammatory cells. The standard sputum processing method was refined to improve sputum cell viability. The flow cytometric assay was used to assess the reproducibility of the measurement of STAT1 phosphorylation and the in vitro activity of a pan JAK-inhibitor on three separate visits in patients with COPD. Results Upregulation of STAT1 phosphorylation was measured following in vitro IFNγ stimulation of sputum macrophages (stimulated/unstimulated ratio 1.57; p<0.00001). Upregulation was inhibited following in vitro preincubation with a pan JAK-inhibitor (inhibited+stimulated/unstimulated ratio 0.97). STAT1 phosphorylation activity could only be measured in macrophages. Conclusions Sputum from patients with COPD can be used to reproducibly measure phospho-STAT expression in sputum macrophages. The flow cytometry-based method can be used to evaluate kinase inhibitors in vitro and subsequently in ex vivo studies. The assay is particularly useful for the assessment of inhaled compounds where whole blood assays may not be relevant. PMID:27403320

  1. Expanding the potential of standard flow cytometry by extracting fluorescence lifetimes from cytometric pulse shifts

    PubMed Central

    Cao, Ruofan; Naivar, Mark A; Wilder, Mark; Houston, Jessica P

    2014-01-01

    Fluorescence lifetime measurements provide information about the fluorescence relaxation, or intensity decay, of organic fluorophores, fluorescent proteins, and other inorganic molecules that fluoresce. The fluorescence lifetime is emerging in flow cytometry and is helpful in a variety of multiparametric, single cell measurements because it is not impacted by nonlinearity that can occur with fluorescence intensity measurements. Yet time-resolved cytometry systems rely on major hardware modifications making the methodology difficult to reproduce. The motivation of this work is, by taking advantage of the dynamic nature of flow cytometry sample detection and applying digital signal processing methods, to measure fluorescence lifetimes using an unmodified flow cytometer. We collect a new lifetime-dependent parameter, referred to herein as the fluorescence-pulse-delay (FPD), and prove it is a valid representation of the average fluorescence lifetime. To verify we generated cytometric pulses in simulation, with light emitting diode (LED) pulsation, and with true fluorescence measurements of cells and microspheres. Each pulse is digitized and used in algorithms to extract an average fluorescence lifetime inherent in the signal. A range of fluorescence lifetimes is measurable with this approach including standard organic fluorophore lifetimes (∼1 to 22 ns) as well as small, simulated shifts (0.1 ns) under standard conditions (reported herein). This contribution demonstrates how digital data acquisition and signal processing can reveal time-dependent information foreshadowing the exploitation of full waveform analysis for quantification of similar photo-physical events within single cells. © 2014 The Authors. Published by Wiley Periodicals, Inc. PMID:25274073

  2. Prospects and limits of the flow cytometric seed screen – insights from Potentilla sensu lato (Potentilleae, Rosaceae)

    PubMed Central

    Dobeš, Christoph; Lückl, Andrea; Hülber, Karl; Paule, Juraj

    2013-01-01

    The flow cytometric seed screen allows for identification of reproductive modes of seed formation and inference of the ploidy of contributing gametes. However, the lack of a mathematical formalization to infer male/female genomic contributions, and the prerequisite of a binucleate female contribution to the endosperm limits its applicability. We evaluated this assumption combining a DNA-based progeny survey with a comparison of the cytology of reproductive pathways co-occurring within single individuals representing 14 Potentilleae species from six phylogenetic lineages. A numerical framework valid for sexual and pseudogamous taxa was developed, enabling quantification of female and male genomes contributing to embryo and endosperm independent of gametophyte origins, numbers of sperm involved and ploidy of parents. The inference strongly depended on accurate peak index estimation. The endosperm of Potentilleae species received a binucleate female contribution in five evolutionary lineages whereas endosperm formation remained uncertain in the Tormentillae. A modified flow cytometric seed screen protocol was developed to cope with low endosperm contents. Evolutionary conservation of a binucleate female contribution to the endosperm suggested wide applicability of flow cytometric seed screen – at least in the Potentilleae. However, alternative progeny surveys and precise embryo/endosperm ploidy estimates are required for a comprehensive understanding of the cytology of seed formation. PMID:23425259

  3. Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin- or fluorescein-labeled dUTP

    SciTech Connect

    Yang, Gang; Olson, J.C.; Pu, R.; Vyas, G.N.

    1995-10-01

    Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type-1 (HrV-1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein-12-dUTP (fluo-dUTP) or digoxigenin-11-dUTP (dig-dUTP) into the PCR-amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin-coated beads which were finally analyzed in a flow cytometer by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo-dUTP and (2) immunodetection of the amplicons incorporating dig-dUTP by anti-digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV-1 proviral DNA sequences, the immunodetection of dig-dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12-14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV-1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig-dUTP incorporation in amplicons, hybridization with a pair of sense-antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR-diagnosis of HIV-1 infection. 21 refs., 2 figs., 4 tabs.

  4. Genome Size Variation among and within Camellia Species by Using Flow Cytometric Analysis

    PubMed Central

    Zhang, Qun-Jie; Gao, Li-Zhi

    2013-01-01

    Background The genus Camellia, belonging to the family Theaceae, is economically important group in flowering plants. Frequent interspecific hybridization together with polyploidization has made them become taxonomically “difficult taxa”. The DNA content is often used to measure genome size variation and has largely advanced our understanding of plant evolution and genome variation. The goals of this study were to investigate patterns of interspecific and intraspecific variation of DNA contents and further explore genome size evolution in a phylogenetic context of the genus. Methodology/Principal Findings The DNA amount in the genus was determined by using propidium iodide flow cytometry analysis for a total of 139 individual plants representing almost all sections of the two subgenera, Camellia and Thea. An improved WPB buffer was proven to be suitable for the Camellia species, which was able to counteract the negative effects of secondary metabolite and generated high-quality results with low coefficient of variation values (CV) <5%. Our results showed trivial effects on different tissues of flowers, leaves and buds as well as cytosolic compounds on the estimation of DNA amount. The DNA content of C. sinensis var. assamica was estimated to be 1C = 3.01 pg by flow cytometric analysis, which is equal to a genome size of about 2940 Mb. Conclusion Intraspecific and interspecific variations were observed in the genus Camellia, and as expected, the latter was larger than the former. Our study suggests a directional trend of increasing genome size in the genus Camellia probably owing to the frequent polyploidization events. PMID:23724111

  5. Flow cytometric analysis of circulating platelet-monocyte aggregates in whole blood: methodological considerations.

    PubMed

    Harding, Scott A; Din, Jehangir N; Sarma, Jaydeep; Jessop, Alasdair; Weatherall, Mark; Fox, Keith A A; Newby, David E

    2007-08-01

    Platelet-monocyte aggregates are increasingly being used to quantify platelet activation. The variables that influence platelet-monocyte aggregates have not been well defined. We sought to determine the effect of blood collection, handling and processing techniques on detected levels of platelet-monocyte aggregates using a flow cytometric assay. Whole blood was labelled with anti-CD14-PE and anti-CD42a-FITC. Thereafter, samples were fixed and red cells lysed. Analysis was performed with the flow cytometer initially triggering on light scatter and then on FL-2 to identify CD14-PE positive monocytes. Platelet-monocyte aggregates were defined as monocytes positive for CD42a. The effect of collection, handling and processing techniques on this assay were assessed. Anticoagulation with heparin (20.1 +/- 2.0%), PPACK (16.8 +/- 1.9%), sodium citrate (12.3 +/- 1.6%) and EDTA (9.5 +/- 1.0%) resulted in markedly different levels of platelet-monocyte aggregation (P < 0.0001). Platelet-monocyte aggregation was higher in samples obtained from intravenous cannulae compared to those obtained by venepuncture (20.9 +/- 3.9% vs.13.8 +/- 2.4%, P = 0.03). For every 10 minutes of delay prior to processing platelet-monocyte aggregates increased by 2.8% (P = 0.0001) in PPACK anticoagulated blood and 1.7% (P = 0.01) in citrate anticoagulated blood. Erythrocyte lysis together with fixation does not affect platelet-monocyte aggregation. Platelet-monocyte aggregates remained stable over 24 hours when fixed and stored at 4 degrees C. Multiple handling and processing factors may affect platelet-monocyte aggregation. We recommend the measurement of platelet-monocyte aggregates on samples collected by direct venepuncture, using a direct thrombin inhibitor as the anticoagulant and minimising the time delay before sample fixation. PMID:17721630

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

    USGS Publications Warehouse

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

    2004-01-01

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

  7. A histological and flow cytometric study of dog brain endothelial cell injuries in delayed radiation necrosis

    SciTech Connect

    Yamaguchi, N.; Yamashima, T.; Yamashita, J. )

    1991-04-01

    The pathogenesis of delayed cerebral radiation necrosis was studied histologically and biochemically in 25 dogs with special attention to vascular endothelial cell injuries. The dogs were sacrificed 3 to 30 months after irradiation with a single dose of 15 Gy to the head. Brain specimens were appropriately fixed for light and electron microscopic studies, and capillary endothelial cells were isolated for flow cytometric study. The endothelial cells were stained with acridine orange, then the cell ratios in the reproductive phase (S + G2 + M) were investigated with flow cytometry. Thereafter, Feulgen hydrolysis and computer analysis of the hydrolysis curves were performed to examine the qualitative changes in deoxyribonucleic acid (DNA) of endothelial cells after irradiation. Under light microscopy, spongy degeneration with small cell infiltration was observed, especially in the frontal white matter, at 6 months after irradiation. At 9 months, necrotic foci appeared and developed until 15 months after irradiation. Blood vessels around the necrotic area showed luminal narrowing with endothelial hyperplasia and proliferation. At 30 months, no fresh necrotic lesions were observed. Under electron microscopy, endothelial cells of capillaries and small vessels around the necrotic area showed an increase of pinocytosis, and in the nuclei there was an increase of infoldings and euchromatin. The cell ratios in the reproductive phase were 14.5% to 23.3% (maximum at 9 months) in the irradiated group compared to 6.4% in the control group. The rate constant of apurinic acid production, a parameter correlating with DNA transcriptional activity, was minimum at 3 months and maximum at 9 months after irradiation. The data suggest that impairment of the microcirculation plays an important role in the pathogenesis of delayed radiation necrosis.

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

    PubMed

    Gauthier, Julie D; Jenkins, Jill A; La Peyre, Jerome F

    2004-06-01

    Parasite surface glycoconjugates are frequently involved in cellular recognition and colonization of the host. This study reports on the identification of Perkinsus marinus surface carbohydrates by flow cytometric analyses of fluorescein isothiocyanate-conjugated lectin binding. Lectin-binding specificity was confirmed by sugar inhibition and Kolmogorov-Smirnov statistics. Clear, measurable fluorescence peaks were discriminated, and no parasite autofluorescence was observed. Parasites (GTLA-5 and Perkinsus-1 strains) harvested during log and stationary phases of growth in a protein-free medium reacted strongly with concanavalin A and wheat germ agglutinin, which bind to glucose-mannose and N-acetyl-D-glucosamine (GlcNAc) moieties, respectively. Both P. marinus strains bound with lower intensity to Maclura pomifera agglutinin, Bauhinia purpurea agglutinin, soybean agglutinin (N-acetyl-D-galactosamine-specific lectins), peanut agglutinin (PNA) (terminal galactose specific), and Griffonia simplicifolia II (GlcNAc specific). Only background fluorescence levels were detected with Ulex europaeus agglutinin I (L-fucose specific) and Limulus polyphemus agglutinin (sialic acid specific). The lectin-binding profiles were similar for the 2 strains except for a greater relative binding intensity of PNA for Perkinsus-1 and an overall greater lectin-binding capacity of Perkinsus-1 compared with GTLA-5. Growth stage comparisons revealed increased lectin-binding intensities during stationary phase compared with log phase of growth. This is the first report of the identification of surface glycoconjugates on a Perkinsus spp. by flow cytometry and the first to demonstrate that differential surface sugar expression is growth phase and strain dependent. PMID:15270084

  9. Flow cytometric characterization of rat thymus cells in a radiation-dominated model of combined injury. Scientific report

    SciTech Connect

    Kaffenberger, K.; Gruber, D.F.; MacVittie, T.J.

    1988-01-01

    Thymuses of rats that had been: (a) gamma-irradiated 500 cGy whole-body radiation (R), or (b) thermally injured 20% BSA dorsal, scald burn (TI), or c) combined injured irradiation followed by burn (CI) were studied for involution and recovery processes after sublethal treatments. The expression of surface antigens on thymic cells before and after injuries was evaluated using the monoclonal antibodies and flow cytometric analysis. Thymic cellularity decreased to less than 1% of normal (N), age-matched rats by 4 days after R or CI. Recovery reached 60% to 70% of N by 28 days post treatments. Expression of OX7, OX8, W3/13, and W3/25 antigens all reached nadirs of 40% of N by day 4 after R and CI. Recovery of antigen expression, except for W3/25, was near completion by day 7 after R and CI. Changes in antigen expression after TI were less pronounced for all mcAB tested. Variations in relative labeling of nonlymphoid thymic cells with OX4 (Ia-antigen) reflected the disappearance and recovery of radiosensitive lymphoid thymocytes. The similarity of results after R and CI demonstrate that the model of CI is radiation-dominated. The addition of burn injury to radiation trauma had no synergistically damaging effect on the parameters studied.

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

    SciTech Connect

    Jensen, R.H.; Bigbee, W.L.; Langlois, R.G.; Grant, S.G. ); Pleshanov, P.G. ); Chirkov, A.A. ); Pilinskaya, M.A. )

    1990-09-12

    An analytical technique has been developed that allows laser-based flow cytometric measurement of the frequency of red blood cells that have lost allele-specific expression of a cell surface antigen due to genetic toxicity in bone marrow precursor cells. Previous studies demonstrated a correlation of such effects with the exposure of each individual to mutagenic phenomena, such as ionizing radiation, and the effects can persist for the lifetime of each individual. During the emergency response to the nuclear power plant accident at Chernobyl, Ukraine, USSR, a number of people were exposed to whole body doses of ionizing radiation. Some of these individuals were tested with this laser-based assay and found to express a dose-dependent increase in the frequency of variant red blood cells that appears to be a persistent biological effect. All data indicate that this assay might well be used as a biodosimeter to estimate radiation dose and also as an element to be used for estimating the risk of each individual to develop cancer due to radiation exposure. 17 refs., 5 figs.

  11. Excimer fluorescence compared to depolarization in the flow cytometric characterization of lateral membrane mobility in platelets

    NASA Astrophysics Data System (ADS)

    Rothe, Gregor; Schaefer, Buerk; Wimmer, Martin S.; Schmitz, Gerd

    1998-04-01

    An altered cellular membrane fluidity secondary to changes of cholesterol metabolism is a potentially important mechanism in the pathogenesis of atherosclerosis. Especially in blood platelets an increased sensitivity for stimulation dependent aggregation which is a risk factor for thrombosis has been experimentally linked to disorders of lipid and lipoprotein metabolism. The goal of this study was the development of a flow cytometric assay for the direct analysis of cellular membrane microviscosity in correlation to activation associated phenotypic changes of platelets in vitro. The analysis of fluorescence polarization following the staining of hydrophobic lipid regions of cell membranes with the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) is a well established method for the analysis of membrane fluidity. The extent of fluorescence anisotropy dependent on the rotational mobility of this fluorochrome is indirectly proportional to the microviscosity of the stained membrane subcompartment. In this study, an alternative and more simple method based on the diffusion dependent excimer formation of pyrenedecanoic acid (PDA) (J. Immunol. Methods 96:225-31, 1987) was characterized in comparison to the DPH method as a reference. Human platelets showed a rapid uptake of both DPH and PDA resulting in the staining primarily of the plasma membrane after up to 30 min of incubation. Staining analyzed at 351 nm excitation resulted in a saturation of the depolarization coefficient of DPH at 20 (mu) M but an increase of the excimer to monomer ratio of PDA with increasing dye concentration. A 'membrane fluidity coefficient' which saturated at 5 (mu) M PDA was calculated as the excimer fluorescence divided through the square of monomer fluorescence thereby correcting for the influence of dye concentration on excimer formation. The temperature dependent changes of membrane viscosity were further used as a model for the comparison of both methods. Cells analyzed at temperatures

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

    PubMed

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

    2016-01-01

    Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz(®) solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high. PMID:27379043

  13. A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

    PubMed Central

    Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J.; Haynes, Mark K.; Bologa, Cristian G.; Oprea, Tudor I.; Tegos, George P.; Sklar, Larry A.; Krishnamurthy, Partha

    2012-01-01

    ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity. PMID:22808084

  14. Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa.

    PubMed

    Parrilla, Inmaculada; Vazquez, Juan M; Gil, Maria A; Caballero, Ignacio; Almiñana, Carmen; Roca, Jordi; Martinez, Emilio A

    2005-07-01

    Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h). PMID:15935845

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

    PubMed Central

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

    2016-01-01

    Bacterial abundance is a fundamental measure in microbiology, but its assessment is often tedious, especially for soil, and sediment samples. To overcome this limitation, we adopted a time-efficient flow-cytometric (FCM) counting method involving cell detachment and separation from matrix particles by centrifugation in tubes receiving sample suspensions and Histodenz® solution. We used this approach to assess bacterial abundances in diverse soils (natural and agricultural), sediments (streams and lakes) and sludge from sand-filters in a drinking water treatment plant and compared the results to bacterial abundances determined by two established methods, epifluorescence microscopy (EM) and adenosine triphosphate (ATP) quantification. Cell abundances determined by FCM and EM correlated fairly well, although absolute cell abundances were generally lower when determined by FCM. FCM also showed significant relations with cell counts converted from ATP concentrations, although estimates derived from ATP determinations were typically higher, indicating the presence of ATP sources other than bacteria. Soil and sediment organic matter (OM) content influenced the goodness of fit between counts obtained with EM and FCM. In particular, bacterial abundance determined by FCM in samples containing less than 10% OM, such as stream sediment, was particularly well correlated with the cell counts assessed by EM. Overall, these results suggest that FCM following cell detachment and purification is a useful approach to increase sample throughput for determining bacterial abundances in soils, sediments and sludge. However, notable scatter and only partial concordance among the FCM and reference methods suggests that protocols require further improvement for assessments requiring high precision, especially when OM contents in samples are high. PMID:27379043

  16. Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification.

    PubMed

    Rosseau, S; Seeger, W; Pralle, H; Lohmeyer, J

    1994-08-01

    The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8074245

  17. FLOW CYTOMETRIC DETECTION AND SIZING OF FLUORESCENT PARTICLES DEPOSITED AT A SEWAGE OUTFALL SITE

    EPA Science Inventory

    A suspension of fluorescent pigment particles (total mass, 120 kg) was injected over a period of several hours into a sewage outfall discharging into Salem Sound, MA. low cytometric analysis was successfully used to identify, quantify, and size the fluorescent pigment particles i...

  18. Automated classification of patients with chronic lymphocytic leukemia and immunocytoma from flow cytometric three-color immunophenotypes.

    PubMed

    Valet, G K; Höffkes, H G

    1997-12-15

    The goal of this study was the discrimination between chronic lymphocytic leukemia (B-CLL), clinically more aggressive lymphoplasmocytoid immunocytoma (LP-IC) and other low-grade non-Hodgkin's lymphomas (NHL) of the B-cell type by automated analysis of flow cytometric immunophenotypes CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5 and CD10/23/19 from peripheral blood and bone marrow aspirate leukocytes using the multiparameter classification program CLASSIF1. The immunophenotype list mode files were exhaustively evaluated by combined lymphocyte, monocyte, and granulocyte (LMG) analysis. The results were introduced into databases and automatically classified in a standardized way. The resulting triple matrix classifiers are laboratory and instrument independent, error tolerant, and robust in the classification of unknown test samples. Practically 100% correct individual patient classification was achievable, and most manually unclassifiable patients were unambiguously classified. It is of interest that the single lambda/CD19/5 antibody triplet provided practically the same information as the full set of the five antibody triplets. This demonstrates that standardized classification can be used to optimize immunophenotype panels. On-line classification of test samples is accessible on the Internet: http://www.biochem.mpg.de/valet/leukaem1.html Immunophenotype panels are usually devised for the detection of the frequency of abnormal cell populations. As shown by computer classification, most the highly discriminant information is, however, not contained in percentage frequency values of cell populations, but rather in total antibody binding, antibody binding ratios, and relative antibody surface density parameters of various lymphocyte, monocyte, and granulocyte cell populations. PMID:9440819

  19. Flow Cytometric Analysis of Protective T-Cell Response Against Pulmonary Coccidioides Infection.

    PubMed

    Hung, Chiung-Yu; Wozniak, Karen L; Cole, Garry T

    2016-01-01

    The incidence of systemic fungal infections has increased throughout the world, spurring much interest in developing effective vaccines. Coccidioidomycosis, also known as San Joaquin Valley fever, is a potentially life-threatening respiratory mycosis. A vaccine against Coccidioides infection would contribute significantly to the well-being of the approx. 30 million residents in the Southwestern USA as well as the multitude of travelers who annually visit the endemic regions. We have applied a live, attenuated vaccine (∆T) to explore the nature of vaccine immunity in mice after intranasal challenge with a potentially lethal dose of Coccidioides spores. Coccidioides spores are airborne and highly infectious for mammalian hosts and classified as a biosafety level 3 agent. T cells are critical in the development of protective immunity against a variety of microorganisms as well as the development of autoimmune disease and allergic responses. Profiles of cytokines detected in lung homogenates of ∆T-vaccinated mice were indicative of a mixed Th1, Th2, and Th17 immune response. We have developed an intracellular cytokine staining and flow cytometric (ICS) technique to measure activated CD4(+) and CD8(+) T cells and IFN-γ-, IL-4-, IL-5-, and IL-17A-producing T cells in the lungs of mice that are challenged with a potentially lethal dose of Coccidioides spores. The numbers of pulmonary Th1 and Th17 cells during the first 2 weeks post-challenge showed a progressive increase in vaccinated mice and corresponded with reduction of fungal burden. In this protocol, we describe the methodology for culture and isolation of the live, attenuated ΔT spores of Coccidioides used to vaccinate mice, preparation of pulmonary cells, and staining protocol for cell surface markers and intracellular cytokines. This is the most reliable and robust procedure to measure frequencies and numbers of each selected T-cell subsets in lungs of vaccinated versus control mice and can be readily

  20. Proliferation markers Ki-67 and p105 in soft-tissue lesions. Correlation with DNA flow cytometric characteristics.

    PubMed Central

    Swanson, S. A.; Brooks, J. J.

    1990-01-01

    Frozen tissue immunoreactivity with Ki-67, a monoclonal antibody that recognizes a nuclear antigen in nonresting or proliferating cells, was compared to DNA flow cytometry results (from fresh tissue) in a diverse group of 60 soft-tissue lesions. Both DNA index and Ki-67 score were independently reported to be associated with grade and prognosis in sarcomas, but no direct comparison of these two variables was made. It was attempted to measure proliferative activity in fixed paraffin-embedded tissues immunohistochemically in a subset of lesions using an antibody to another nuclear proliferation antigen, p105. Lesions were given a grade according to lesion category (reactive, 1; benign, 2; low-grade malignant, 3; and high-grade malignant, 4). Ki-67 reactivity correlated relatively well with this grading system (r = 0.59); benign lesions usually exhibited a low Ki-67 score and malignant lesions usually but not always exhibited a high score. For example, some malignant fibrous histiocytomas contained only rare positive cells. Some disparity between Ki-67 score and grade and within histologic types indicates some independence from these features, a fact that may be important when correlation with prognosis is performed. However Ki-67 did not correlate well with flow data such as percentage S phase (r = 0.30), percentage S + G2M phases (r = 0.37), or DNA index (r = 0.39). This probably is due to the fact that Ki-67 also marks cells in the G1 phase, whereas these are excluded in flow data analyses. Anti-p105 highlighted almost all nuclei in all cases tested, including fibromatosis, and did not correlate with Ki-67 score, histologic grade or DNA flow cytometric data. Results with p105 could not be favorably affected by titration experiments. It is reasonable to conclude that the Ki-67 score is a variable related to but independent of histologic grade, histologic type, and DNA flow values. Whether it is prognostically important in human sarcomas, as has been suggested

  1. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    SciTech Connect

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. )

    1990-08-15

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

  2. Flow cytometric and radioisotopic determinations of platelet survival time in normal cats and feline leukemia virus-infected cats

    SciTech Connect

    Jacobs, R.M.; Boyce, J.T.; Kociba, G.J.

    1986-01-01

    This study demonstrates the potential usefulness of a flow cytometric technique to measure platelet survival time in cats utilizing autologous platelets labeled in vitro with fluorescein isothiocyanate (FITC). When compared with a 51Cr method, no significant differences in estimated survival times were found. Both the 51Cr and FITC-labeling procedures induced similar changes in platelet shape and collagen-induced aggregation. Platelets labeled with FITC had significantly greater volumes compared with those of glutaraldehyde-fixed platelets. These changes were primarily related to the platelet centrifugation and washing procedures rather than the labels themselves. This novel technique potentially has wide applicability to cell circulation time studies as flow cytometry equipment becomes more readily available. Problems with the technique are discussed. In a preliminary study of the platelet survival time in feline leukemia virus (FeLV)-infected cats, two of three cats had significantly reduced survival times using both flow cytometric and radioisotopic methods. These data suggest increased platelet turnover in FeLV-infected cats.

  3. Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots.

    PubMed

    Staszkiewicz, Jaroslaw; Gimble, Jeffrey; Cain, Courtney; Dietrich, Marilyn; Burk, David; Kirk-Ballard, Heather; Gawronska-Kozak, Barbara

    2009-01-16

    This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). PMID:19056354

  4. Flow cytometric and immunohistochemical detection of in vivo BrdU-labeled cells in mouse fat depots

    PubMed Central

    Staszkiewicz, Jaroslaw; Gimble, Jeffrey; Cain, Courtney; Dietrich, Marilyn; Burk, David; Kirk-Ballard, Heather; Gawronska-Kozak, Barbara

    2009-01-01

    This study has determined the natural frequency and localization of progenitor/stem cells within fat depots in situ based on their ability to retain DNA nucleotide label (BrdU). Neonate and mature male C57BL6/J mice were injected intraperitoneally with BrdU- and label-retaining cells (LRC) were quantified in fat depots by immunohistochemical, immunofluorescent, and flow cytometric methods. In neonates, LRC constituted 27% of the cells in inguinal fat (iWAT) and 65% in interscapular brown fat (BAT) after Day 10 and 26% of the cells in epididymal fat (eWAT) after Day 28. After 52 days, the LRC accounted for 0.72% of iWAT, 0.53% of eWAT and 1.05% of BAT, respectively. The BrdU-labeled cells localized to two areas: single cells distributed among adipocytes or those adjacent to the blood vessels wall. In mature C57BL6/J mice, flow cytometric analysis determined that a majority of the LRC were also positive for stem cell antigen-1 (Sca-1). PMID:19056354

  5. Comparison of drug release from liquid crystalline monoolein dispersions and solid lipid nanoparticles using a flow cytometric technique

    PubMed Central

    Dawoud, Mohamed Z.; Nasr, Mohamed

    2016-01-01

    Colloidal lipid particles such as solid lipid nanoparticles and liquid crystalline nanoparticles have great opportunities as drug carriers especially for lipophilic drugs intended for intravenous administration. In order to evaluate drug release from these nanoparticles and determine their behavior after administration, emulsion droplets were used as a lipophilic compartment to which the transfer of a model drug was measured. The detection of the model drug transferred from monoolein cubic particles and trimyristin solid lipid nanoparticles into emulsion droplets was performed using a flow cytometric technique. A higher rate and amount of porphyrin transfer from the solid lipid nanoparticles compared to the monoolein cubic particles was observed. This difference might be attributed to the formation of a highly ordered particle which leads to the expulsion of drug to the surface of the crystalline particle. Furthermore, the sponge-like structure of the monoolein cubic particles decreases the rate and amount of drug transferred. In conclusion, the flow cytometric technique is a suitable technique to study drug transfer from these carriers to large lipophilic acceptors. Monoolein cubic particles with their unique structure can be used successfully as a drug carrier with slow drug release compared with trimyristin nanoparticles. PMID:27006901

  6. Flow cytometric enumeration of bacterial in the coral surface mucus layer.

    PubMed

    Bettarel, Yvan; Thanh, Mai Chi; Patrice, Got; Antoinette, Adingra; Nadège, Kouadio-Ngbesso; Bui, Van Ngoc; Thierry, Bouvier

    2016-09-01

    The direct counts of bacteria inhabiting coral mucus were performed by flow cytometry testing four fluorescent dyes (SYBR®Green I, HCS, TOPRO®3, SYTO®62) with three different scleractinian species. Results obtained with SYTO62 were the most reliable based on the comparison with standardized epifluorescence counts and the resolution of cytograms. PMID:27302040

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

    PubMed Central

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

    2016-01-01

    Flow cytometry is used extensively to examine immune cells in non-lymphoid tissues. However, a method of flow cytometric analysis that is both comprehensive and widely applicable has not been described. We developed a protocol for the flow cytometric analysis of non-lymphoid tissues, including methods of tissue preparation, a 10-fluorochrome panel for cell staining, and a standardized gating strategy, that allows the simultaneous identification and quantification of all major immune cell types in a variety of normal and inflamed non-lymphoid tissues. We demonstrate that our basic protocol minimizes cell loss, reliably distinguishes macrophages from dendritic cells (DC), and identifies all major granulocytic and mononuclear phagocytic cell types. This protocol is able to accurately quantify 11 distinct immune cell types, including T cells, B cells, NK cells, neutrophils, eosinophils, inflammatory monocytes, resident monocytes, alveolar macrophages, resident/interstitial macrophages, CD11b- DC, and CD11b+ DC, in normal lung, heart, liver, kidney, intestine, skin, eyes, and mammary gland. We also characterized the expression patterns of several commonly used myeloid and macrophage markers. This basic protocol can be expanded to identify additional cell types such as mast cells, basophils, and plasmacytoid DC, or perform detailed phenotyping of specific cell types. In examining models of primary and metastatic mammary tumors, this protocol allowed the identification of several distinct tumor associated macrophage phenotypes, the appearance of which was highly specific to individual tumor cell lines. This protocol provides a valuable tool to examine immune cell repertoires and follow immune responses in a wide variety of tissues and experimental conditions. PMID:26938654

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

    PubMed

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

    2015-02-01

    Cytometric sorting of frozen-thawed sperm can overcome difficulties caused by the unavailability of sorting facilities on farms where semen is collected from male livestock. In order to optimize the cytometric sex-sorting procedure, effects of Hoechst33342 staining on the viability and cytometric sorting efficiency of frozen-thawed ram sperm were evaluated. The frozen-thawed sperm were stained with Hoechst33342 at various dye concentrations (80 μM, 120 μM, 160 μM, 200 μM, 240 μM, or 320 μM) for 45 min to evaluate effects of dye dose. The frozen-thawed sperm were stained with 160 μM Hoechst33342 for various durations (0 min, 15 min, 30 min, 45 min, 60 min, 75 min, or 90 min) to evaluate effects of staining duration. Sperm motility and moving velocity were analyzed using a computer-assisted sperm analysis system (CASAS). Acrosome status, membrane integrity, and distribution of phosphatidylserine (PS) in Hoechst33342-stained sperm were analyzed using flow cytometry after staining with fluorescein isothiocyanate-labeled lectin from pisum sativum (FITC-PSA), Annexin V, or propidium iodide (PI). The fertility of Hoechst33342-stained sperm was analyzed by in vitro fertilization (IVF). A high-speed cell sorter was used to evaluate effects of Hoechst33342 staining on cytometric sex-sorting of frozen-thawed sperm. The motility, moving velocity, membrane integrity, and PS distribution of Hoechst33342-stained sperm were significantly different from that of immediately thawed sperm (P<0.05). However, there is no significant difference existing among the Hoechst33342-stained groups with respect to the above evaluated parameters. Additionally, along with the staining durations, the adverse effects of the staining procedure on sperm showed a steady increase. However, Hoechst33342 staining did not damage acrosome and in vitro fertilizing capability of frozen-thawed ram sperm. Results of cytometric sorting indicated that frozen-thawed sperm can be efficiently sorted into two

  9. Flow cytometric two-color staining technique for simultaneous determination of human erythrocyte membrane antigen and intracellular malarial DNA.

    PubMed

    Pattanapanyasat, K; Webster, H K; Udomsangpetch, R; Wanachiwanawin, W; Yongvanitchit, K

    1992-01-01

    A novel fixative and permeabilization method is described which allows simultaneous flow cytometric detection of red blood cell membrane antigen and intracellular malaria parasites. To illustrate the method, red blood cells from patients with paroxysmal nocturnal hemoglobinuria were infected with Plasmodium falciparum and maintained in synchronous red blood cell culture. The infected red blood cells were immunolabeled with antibodies directed to the complement regulatory protein decay-accelerating factor (DAF) followed by subsequent fixations in paraformaldehyde and then glutaraldehyde in phosphate-buffered saline. Finally, DNA of the intraerythrocytic parasites was stained with propidium iodide. Using this technique, cellular morphology was well preserved, no cell aggregation was observed, and high-quality indirect immunofluorescence and parasite DNA staining were obtained with negligible nonspecific labelling. Simultaneous measurement of parasite DNA and red blood cell membrane determinants makes possible the investigation of alterations of red cell membrane proteins in association with development of intracellular malaria parasites. PMID:1372210

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

    PubMed

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

    2016-02-16

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

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

    PubMed

    Buys, Angela; Macdonald, Raynard; Crafford, Jannie; Theron, Jacques

    2014-01-01

    Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT). Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA) was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA) and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA) tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL). These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required. PMID:24832497

  12. A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to Nitrite Reduction by Deoxygenated Erythrocytes

    PubMed Central

    Akrawinthawong, Krittapoom; Park, Ji Won; Piknova, Barbora; Sibmooh, Nathawut; Fucharoen, Suthat; Schechter, Alan N.

    2014-01-01

    Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects. PMID:24642865

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

    PubMed Central

    Fröhling, Antje; Schlüter, Oliver

    2015-01-01

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

  14. High frequency of circulating HBcAg-specific CD8 T cells in hepatitis B infection: a flow cytometric analysis

    PubMed Central

    Matsumura, S; Yamamoto, K; Shimada, N; Okano, N; Okamoto, R; Suzuki, T; Hakoda, T; Mizuno, M; Higashi, T; Tsuji, T

    2001-01-01

    Viral antigen-specific T cells are important for virus elimination. We studied the hepatitis B virus (HBV)-specific T cell response using flow cytometry. Three phases of HBV infection were studied: Group A, HBeAg (+) chronic hepatitis; Group B, HBeAb (+) HBV carrier after seroconversion; and Group C, HBsAb (+) phase. Peripheral T cells were incubated with recombinant HB core antigen (HBcAg), and intracytoplasmic cytokines were analysed by flow cytometry. HBcAg-specific CD4 and CD8 T cells were identified in all three groups and the number of IFN-γpositive T cells was greater than TNF-α-positive T cells. The frequency of IFN-γ-positive CD4 and CD8 T cells was highest in Group C, compared with Groups A and B. No significant difference in the HBcAg-specific T cell response was observed between Group A and Group B. The HBcAg-specific CD8 T cell response was diminished by CD4 depletion, addition of antibody against human leucocyte antigen (HLA) class I, class II or CD40L. Cytokine-positive CD8 T cells without HBcAg stimulation were present at a high frequency (7 of 13 cases) in Group B, but were rare in other groups. HBcAg-specific T cells can be detected at high frequency by a sensitive flow cytometric analysis, and these cells are important for controlling HBV replication. PMID:11472405

  15. Optimal cellular preservation for high dimensional flow cytometric analysis of multicentre trials.

    PubMed

    Ng, Amanda A P; Lee, Bernett T K; Teo, Timothy S Y; Poidinger, Michael; Connolly, John E

    2012-11-30

    High dimensional flow cytometry is best served by centralized facilities. However, the difficulties around sample processing, storage and shipment make large scale international studies impractical. We therefore sought to identify optimized fixation procedures which fully leverage the analytical capability of high dimensional flow cytometry without the need for complex cell processing or a sustained cold chain. Whole blood staining procedure was employed to investigate the applicability of fixatives including Cyto-Chex® Blood Collection tube (Streck), Transfix® (Cytomark), 1% and 4% paraformaldehyde to centralized analysis of field trial samples. Samples were subjected to environmental conditions which mimic field studies, without refrigerated shipment and analyzed across 10 days, based on cell count and marker expression. This study showed that Cyto-Chex® demonstrated the least variability in absolute cell count relative to samples analyzed directly from donors in the absence of fixation. Transfix® was better at preserving the marker expression among all fixatives. However, Transfix® caused marked increased cell membrane permeabilization and was detrimental to intracellular marker identification. Paraformaldehyde fixation, at either 1% or 4% concentrations, was unfavorable for cell preservation under the conditions tested and thus not recommended. Using these data, we have created an online interactive tool which enables researchers to evaluate the impact of different fixatives on their panel of interest. In this study, we have identified Cyto-Chex® as the optimal cellular preservative for high dimensional flow cytometry in large scale studies for shipped whole blood samples, even in the absence of a sustained cold chain. PMID:22922462

  16. Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

    NASA Astrophysics Data System (ADS)

    Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

    2014-03-01

    Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

  17. Flow cytometric method for the assessment of the minimal inhibitory concentrations of antibacterial agents to Mycoplasma agalactiae.

    PubMed

    Assunção, Patrícia; Antunes, Nuno T; Rosales, Ruben S; de la Fe, Christian; Poveda, Carlos; Poveda, José B; Davey, Hazel M

    2006-10-01

    In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MIC) of seven antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, oxytetracycline, and tylosin) on Mycoplasma (M.) agalactiae. Flow cytometry was able to detect M. agalactiae inhibition from 6 h postincubation, although it seems that definitive MIC values determined by flow cytometry were only possible at 12-h postincubation. However, the results obtained by the traditional method were only obtained at 24 h, when a visible change in the medium had occurred. At 24 h, both methods gave the same result for six antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, and oxytetracycline); whereas flow cytometry gave slightly higher MIC for tylosin. This was attributed to the fact that the M. agalactiae growth that had occurred in the tubes containing tylosin was not enough to visibly change the color of the medium. Futhermore, flow cytometry detected that inhibitory concentrations of oxytetracycline, chloramphenicol, and tylosin as judged at 24 h were not able to inhibit the M. agalactiae growth after 48 h. MIC values of enrofloxacin and ciprofloxacin were sufficient only to maintain the total counts per milliliter throughout the time matched samples, whereas higher concentrations of theses antibacterial agents reduced the total counts per milliliter over the course of the experiment. The main advantage of the flow cytometric method is that MIC results for M. agalactiae can be obtained in a shorter time than is possible with the traditional method. The method presented makes identification of resistant populations of M. agalactiae possible and, unlike the traditional method, allows the effect of each antibacterial agent to be determined in real-time at the single-cell level. PMID:16998868

  18. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

    NASA Astrophysics Data System (ADS)

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-07-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV-) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV- centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

  19. Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating.

    PubMed

    Hui, Yuen Yung; Su, Long-Jyun; Chen, Oliver Yenjyh; Chen, Yit-Tsong; Liu, Tzu-Ming; Chang, Huan-Cheng

    2014-01-01

    Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV(-)) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV(-) centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo. PMID:24994610

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

    PubMed

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

    2016-01-01

    Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)) and non-classical (CD14(+)CD16(++)) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood. PMID:27584036

  1. Screening of promoters from rhizosphere metagenomic DNA using a promoter-trap vector and flow cytometric cell sorting.

    PubMed

    Lee, Se Hee; Kim, Jeong Myeong; Lee, Hyo Jung; Jeon, Che Ok

    2011-02-01

    We constructed a facilitative and efficient promoter-trap vector, pCM-EGFP, for capturing and analyzing functional promoters from environmental DNA. The pCM-EGFP vector showed good chloramphenicol sensitivity and no enhanced green fluorescent protein (EGFP) gene expression. Promoter libraries were constructed for screening promoters responding to naringenin, a key molecule released from plant roots. After electroporation, E. coli transformants were incubated in LB broth containing chloramphenicol (10 μg/ml) to select against transformants with no cloned promoter. E. coli cells were sorted using flow cytometry without naringenin, and then sorted again with high fluorescence after incubation in LB broth with naringenin (1 mM) at 28 °C for 12 h. The inducible properties of approximately 400 sorted cells were evaluated, with most cells showing only strong EGFP gene expression without inducible properties. Two clones (5-4E and 15-3D) displayed naringenin inducibility, and both contained a promoter bounded by a TetR-family regulator. The regulator knock-out mutant of the 5-4E clone lost its ability to be induced by naringenin. In conclusion, the pCM-EGFP vector may be used as an efficient promoter-trap vector and a combination of the vector with flow cytometric cell sorting was demonstrated to be an useful method for screening promoters responding to specific conditions or inducers. PMID:21259288

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

    NASA Astrophysics Data System (ADS)

    Retnakumari, Archana; Jayasimhan, Jasusri; Chandran, Parwathy; Menon, Deepthy; Nair, Shantikumar; Mony, Ullas; Koyakutty, Manzoor

    2011-07-01

    Protein stabilized gold nanoclusters (Au-NCs) are biocompatible, near-infrared (NIR) emitting nanosystems having a wide range of biomedical applications. Here, we report the development of a Au-NC based targeted fluorescent nano-bioprobe for the flow-cytometric detection of acute myeloid leukaemia (AML) cells. Au-NCs with ~ 25-28 atoms showing bright red-NIR fluorescence (600-750 nm) and average size of ~ 0.8 nm were prepared by bovine serum albumin assisted reduction-cum-stabilization in aqueous phase. The protein protected clusters were conjugated with monoclonal antibody against CD33 myeloid antigen, which is overexpressed in ~ 99.2% of the primitive population of AML cells, as confirmed by immunophenotyping using flow cytometry. Au-NC-CD33 conjugates having average size of ~ 12 nm retained bright fluorescence over an extended duration of ~ a year, as the albumin protein protects Au-NCs against degradation. Nanotoxicity studies revealed excellent biocompatibility of Au-NC conjugates, as they showed no adverse effect on the cell viability and inflammatory response. Target specificity of the conjugates for detecting CD33 expressing AML cells (KG1a) in flow cytometry showed specific staining of ~ 95.4% of leukaemia cells within 1-2 h compared to a non-specific uptake of ~ 8.2% in human peripheral blood cells (PBMCs) which are CD33low. The confocal imaging also demonstrated the targeted uptake of CD33 conjugated Au-NCs by leukaemia cells, thus confirming the flow cytometry results. This study demonstrates that novel nano-bioprobes can be developed using protein protected fluorescent nanoclusters of Au for the molecular receptor targeted flow cytometry based detection and imaging of cancer cells.

  3. Flow cytometric analysis of platelet activation under calcium ion-chelating conditions.

    PubMed

    Nishioka, T; Yokota, M; Tsuda, I; Tatsumi, N

    2002-04-01

    Platelet activation and aggregation results in factitious counting and sizing in routine haematology testing. In this study, the possibility of platelet activation in anticoagulated solutions was examined. Whole blood was examined using an automated counter and a flow cytometer before and after strong vortex agitation. Blood treated with ethylenediaminetetraacetic acid (EDTA) exhibited platelet activation both pre- and postagitation but activated platelets did not cause platelet aggregation. With sodium citrate, platelets were only minimally activated both pre- and postagitation. Heparin-treated blood exhibited minimal platelet activation preagitation, but agitation resulted in strong platelet activation and aggregation. Platelet size was increased by agitation in blood with EDTA and with sodium citrate, in association with significant increases in mean platelet volume (MPV) and platelet distribution width (PDW), but MPV and PDW were significantly higher in EDTA solution than in sodium citrate solution. Change in platelet size was observed even in the presence of EDTA, indicating that careful sampling and processing are needed in the collection of specimens. Specimens obtained from patients with EDTA-dependent pseudothrombocytopenia exhibited the same level of activation as controls, although platelets exhibited aggregation in such specimens. In conclusion, platelet activation involving platelet size change can occur in the absence of calcium ions in blood treated with EDTA. PMID:11985558

  4. Flow cytometric lifetime-based cell viability assay using propidium iodide

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Lehnert, Bruce E.; Lehnert, Nancy M.

    1999-05-01

    Assays which discriminate and enumerate dying or dead cells are important in various types of cellular studies. In many instances, there is a need to identify dead cells that interfere with fluorescent probes which are used to measure functional and physiological properties in viable cells. For example, dead cells can introduce analytical errors arising from (1) nonspecific uptake of fluorescent probes, leading to erroneous percentages of positive labeled cells, (2) increased autofluorescence, and (3) altered antigen expression. The ability to detect dead cells is also of importance in determining the effectiveness of cytotoxic agents. Propidium iodide (PPI) exclusion, which is analogous to the non- fluorescent trypan blue dye test for viability, is used extensively in flow cytometry assays. However, the use of PI can potentially limit the application of additional fluorescent probes due to spectral overlap of the probe with PI. In this report we present phase-resolved fluorescence studies on rat and murine thymus cells labeled with phycoerythrin-antiThy 1.1 and phycoerythrin/Texas Red-antiThy 1.2 immunofluorescence markers, respectively, and PI. Overlapping emission spectra are resolved based on differences in fluorescence lifetimes of the probes and PI. These studies demonstrate a new lifetime-based viability method for use in analysis of immunofluorescent probes and for assaying the dynamics of cell killing.

  5. Flow-cytometric analysis of T-lymphocyte subsets in sinistral and dextral patients with gingivitis.

    PubMed

    Orbak, Recep; Canakçi, Varol; Erciyas, Kamile; Kaya, Hasan

    2003-01-01

    The aim of this study was to determine whether there was any change in T-lymphocyte subsets in sinistral and dextral patients with gingivitis. The study was carried out on 36 patients (16 males and 20 females) with gingivitis. The age of the patients ranged from 16 to 25 (mean age = 18.50 +/- 3.85). Patients were divided into two equal groups according to their right or left hand use. Being right- or left-handed was determined with Edinburgh Handedness Inventory (Oldfield). At the beginning of the study, gingival index (GI-Löe-Silness) and plaque index (PI-Silness-Löe) scores were recorded in order to assess the gingival tissue health in patients. At the same time, the biopsy samples were taken from the gingival pocket wall tissues at sites of gingivitis. Then, CD4+ and CD8+ lymphocyte and CD4/CD8 ratio values were determined using flow-cytometry in the biopsy samples. The two groups were compared by using Student's t-test. The normal value in peripheral blood of CD4+ lymphocyte and that of CD8+ lymphocyte are 25-29% and 19-48%, respectively. According to flow cytometry findings, in both sinistrality and dexterity with gingivitis, CD+ and CD8+ lymphocyte values were under the normal value while the CD4/CD8 rate was within normal distribution interval. CD4+ lymphocyte values observed in the sinistral patients were found to be lower than those in the dextral patients. The difference between the CD8+ lymphocyte values in left-handed patients and that obtained in right-handed patients was not found to be statistically significant while the difference between the CD4+ lymphocyte values in left-handed patients and that obtained in right-handed patients was found to be statistically significant (p < .05). In addition, the difference between the CD4/CD8 rate obtained in left-handed patients and that obtained in right-handed patients was found to be statistically very significant (p < .001). Consequently, these findings suggested CD4+ lymphocyte value and CD4/CD8 rate was

  6. Flow cytometric-membrane potential detection of sodium channel active marine toxins: application to ciguatoxins in fish muscle and feasibility of automating saxitoxin detection.

    PubMed

    Manger, Ronald; Woodle, Doug; Berger, Andrew; Dickey, Robert W; Jester, Edward; Yasumoto, Takeshi; Lewis, Richard; Hawryluk, Timothy; Hungerford, James

    2014-01-01

    Ciguatoxins are potent neurotoxins with a significant public health impact. Cytotoxicity assays have allowed the most sensitive means of detection of ciguatoxin-like activity without reliance on mouse bioassays and have been invaluable in studying outbreaks. An improvement of these cell-based assays is presented here in which rapid flow cytometric detection of ciguatoxins and saxitoxins is demonstrated using fluorescent voltage sensitive dyes. A depolarization response can be detected directly due to ciguatoxin alone; however, an approximate 1000-fold increase in sensitivity is observed in the presence of veratridine. These results demonstrate that flow cytometric assessment of ciguatoxins is possible at levels approaching the trace detection limits of our earlier cytotoxicity assays, however, with a significant reduction in analysis time. Preliminary results are also presented for detection of brevetoxins and for automation and throughput improvements to a previously described method for detecting saxitoxins in shellfish extracts. PMID:24830140

  7. Development and Application of Flow-Cytometric Techniques for Analyzing and Sorting Endospore-Forming Clostridia▿ †

    PubMed Central

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

    2008-01-01

    The study of microbial heterogeneity at the single-cell level is a rapidly growing area of research in microbiology and biotechnology due to its significance in pathogenesis, environmental biology, and industrial biotechnologies. However, the tools available for efficiently and precisely probing such heterogeneity are limited for most bacteria. Here we describe the development and application of flow-cytometric (FC) and fluorescence-assisted cell-sorting techniques for the study of endospore-forming bacteria. We show that by combining FC light scattering (LS) with nucleic acid staining, we can discriminate, quantify, and enrich all sporulation-associated morphologies exhibited by the endospore-forming anaerobe Clostridium acetobutylicum. Using FC LS analysis, we quantitatively show that clostridial cultures commonly perform multiple rounds of sporulation and that sporulation is induced earlier by the overexpression of Spo0A, the master regulator of endospore formers. To further demonstrate the power of our approach, we employed FC LS analysis to generate compelling evidence to challenge the long-accepted view in the field that the clostridial cell form is the solvent-forming phenotype. PMID:18931289

  8. Effects of dietary fish oil and vitamin E supplementation on canine lymphocyte proliferation evaluated using a flow cytometric technique.

    PubMed

    LeBlanc, Casey J; Dietrich, Marilyn A; Horohov, David W; Bauer, John E; Hosgood, Giselle; Mauldin, Glenna E

    2007-10-15

    Lymphocyte proliferation and peripheral blood mononuclear cell (PBMC) production of PGE(2) were assayed in 15 healthy dogs fed a basal diet supplemented with either sunflower oil (Group Sunflower oil), sunflower oil and menhaden fish oil (Group Fish oil), or sunflower oil and menhaden fish oil plus alpha-tocopherol acetate for 12 weeks (Group Fish oil + E). Lymphocyte proliferation was determined by a flow cytometric technique utilizing the fluorochrome carboxyfluorescein diacetate succinimidyl ester (CFSE). The PBMC supernatant PGE(2) concentration was assayed using a competitive enzyme-linked immunoassay. Group Fish oil had a significant decrease in lymphocyte proliferation at week 12. PBMC production of PGE(2) was decreased in all three groups but only significantly reduced in groups receiving fish oil supplementation. Based on these results, this level of fish oil supplementation appears to suppress the lymphoproliferative response in healthy, young dogs but this response can be attenuated by high levels of dietary vitamin E supplementation. Furthermore, fish oil-induced reduction in lymphocyte proliferation appears to manifest through a PGE(2)-independent mechanism and is not associated with increased lipid peroxidation. PMID:17658617

  9. Prognostic factors in anal squamous carcinoma: a multivariate analysis of clinical, pathological and flow cytometric parameters in 235 cases.

    PubMed

    Shepherd, N A; Scholefield, J H; Love, S B; England, J; Northover, J M

    1990-06-01

    Clinical, pathological and flow cytometric parameters have been analysed by univariate and multivariate analysis to define those parameters of important prognostic influence in 235 cases of surgically treated squamous carcinoma of the anus and perianal skin. Patients had been treated by anorectal excision (166 patients) or by local excision (69). Analyses were carried out on five data sets--the two surgical subgroups, two groups distinguished by site of tumour and on all 235 patients. Univariate analysis showed many parameters to be of prognostic influence, although histological typing of tumours into the more common histological subtypes was of no prognostic value. Parameters of independent prognostic significance in multivariate analysis were those indicating depth of spread, inguinal lymph node involvement and DNA-ploidy. In this study the subdivision of the rarer types of anal canal tumour, such as mucoepidermoid carcinoma, microcystic squamous carcinoma and small cell anaplastic carcinoma, was relevant confirming that these tumours have a poor prognosis. It is now felt that surgery should not be employed as primary treatment in most cases of anal cancer and the results of this study have to be interpreted with caution when applied to patients treated with radiotherapy with or without chemotherapy. Nevertheless, our findings suggest that the most useful prognostic information can be gleaned from accurate clinical staging and an assessment of DNA-ploidy status. PMID:2376397

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

    PubMed

    Chen, Wenbo; Hasegawa, Daniel K; Arumuganathan, Kathiravetpillai; Simmons, Alvin M; Wintermantel, William M; Fei, Zhangjun; Ling, Kai-Shu

    2015-01-01

    Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1) population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680-690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome. PMID:26463411

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

    PubMed Central

    Chen, Wenbo; Hasegawa, Daniel K.; Arumuganathan, Kathiravetpillai; Simmons, Alvin M.; Wintermantel, William M.; Fei, Zhangjun; Ling, Kai-Shu

    2015-01-01

    Whiteflies of the Bemisia tabaci (Hemiptera: Aleyrodidae) cryptic species complex are among the most important agricultural insect pests in the world. These phloem-feeding insects can colonize over 1000 species of plants worldwide and inflict severe economic losses to crops, mainly through the transmission of pathogenic viruses. Surprisingly, there is very little genomic information about whiteflies. As a starting point to genome sequencing, we report a new estimation of the genome size of the B. tabaci B biotype or Middle East-Asia Minor 1 (MEAM1) population. Using an isogenic whitefly colony with over 6500 haploid male individuals for genomic DNA, three paired-end genomic libraries with insert sizes of ~300 bp, 500 bp and 1 Kb were constructed and sequenced on an Illumina HiSeq 2500 system. A total of ~50 billion base pairs of sequences were obtained from each library. K-mer analysis using these sequences revealed that the genome size of the whitefly was ~682.3 Mb. In addition, the flow cytometric analysis estimated the haploid genome size of the whitefly to be ~690 Mb. Considering the congruency between both estimation methods, we predict the haploid genome size of B. tabaci MEAM1 to be ~680–690 Mb. Our data provide a baseline for ongoing efforts to assemble and annotate the B. tabaci genome. PMID:26463411

  12. Quantitative analysis of cultured thymic reticulo-epithelial cells labelled by different antibodies: a flow cytometric study.

    PubMed Central

    Fabien, N; Auger, C; Bonnard, M; Andreoni, C; Rigal, D; Monier, J C

    1989-01-01

    Quantitative measurements of cultured human and murine thymic, and human thymoma reticuloepithelial cells (REC), immunolabeled by different antibodies (Ab) (TE3, TE4, anti-HTLV p19(p19), lu5, K11 and Aks) and by thymic hormones (thymulin and thymosin alpha 1 (Ta1)) within these cells, were performed using a flow cytometric technique. The anti-keratin polyclonal Ab labeled nearly the whole human or murine population. The p19 monoclonal Ab (MoAb), specific for the subcortical/medullary thymic regions, labelled 37-77% of the human REC. The TE3 MoAb, specific for the cortical region, labelled 54-83% of the REC. These percentages suggest that the cultured thymic REC (TREC) had markers of both regions together and therefore that these markers are not absolutely specific to determine their subcortical/medullary or cortical thymic origin. For the three populations there were more cells containing Ta1 than thymulin. The overlap of the percentage of labelled cells suggests that the same cell could synthesize the two hormones and that these hormones could be localized within the TE3 positive cells. PMID:2649289

  13. Flow-cytometric evaluation of lymphocyte subpopulations in synchronously developing Schistosoma mansoni egg and Sephadex bead pulmonary granulomas.

    PubMed Central

    Remick, D. G.; Chensue, S. W.; Hiserodt, J. C.; Higashi, G. I.; Kunkel, S. L.

    1988-01-01

    Synchronous models of T-cell-mediated and foreign body granulomas were induced in mice by intravenous embolization of Schistosoma mansoni eggs and Sephadex beads, respectively. The authors then performed flow-cytometric analysis of lymphocytes from dispersed granulomas, spleens, and peripheral blood at 4, 8, 16, and 32 days corresponding to the induction, growth, and maintenance, and resolution of these lesions. Lymphocytes were identified on the basis of light scatter characteristics, and the nature of the cells was confirmed by cell sorting and electron-microscopic examination. Lymphocyte subpopulations were characterized with antibodies to lymphocyte surface markers, specifically Ig, Thy 1.2, Lyt 1, Lyt 2, and L3T4. Natural killer cells were identified with anti-asialo GM1. Egg-induced granulomas had more lymphocytes of all phenotypes at all time points. Surprisingly, there was a significant number of cells staining positive for asialo GM1. On Day 16 after embolization there was a greater percentage of helper T cells, as defined by positive staining with L3T4, in the egg model, compared with the bead model. There was no obvious shift of lymphocytes from either the blood or spleen into the granuloma. These data confirm the importance of T cells in the direct participation of granulomatous inflammation, and the large numbers of asialo GM1-positive cells suggest a role for natural killer cells. Images Figure 4 PMID:2451888

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

    PubMed

    Dalgaard, T S; Norup, L R; Pedersen, A R; Handberg, K J; Jørgensen, P H; Juul-Madsen, H R

    2010-06-17

    The objective of this study was to use flow cytometry to assess chicken T cell-mediated immune responses. In this study two inbred genetic chicken lines (L130 and L133) were subjected to two times vaccination against Newcastle disease (ND) and a subsequent challenge by ND virus (NDV) infection. Despite a delayed NDV-specific antibody response to vaccination, L133 appeared to be better protected than L130 in the subsequent infection challenge as determined by the presence of viral genomes. Peripheral blood was analyzed by flow cytometry and responses in vaccinated/challenged birds were studied by 5-color immunophenotyping as well as by measuring the proliferative capacity of NDV-specific T cells after recall stimulation. Immunophenotyping identified L133 as having a significantly lower CD4/CD8 ratio and a lower frequency of gammadelta T cells than L130 in the peripheral T cell compartment. Furthermore, peripheral lymphocytes from L133 exhibited a significantly higher expression of CD44 and CD45 throughout the experiment. Interestingly, also vaccine-induced differences were observed in L133 as immune chickens had a significantly higher CD45 expression on their lymphocytes than the naïve controls. Immune chickens from both lines had a significantly higher frequency of circulating gammadelta T cells than the naïve controls both after vaccination and challenge. Finally, the proliferative capacity of peripheral CD4+ and CD8+ cells specific for NDV was addressed 3 weeks after vaccination and 1 week after infection and found to be significantly higher in L133 than in L130 at both sampling times. In conclusion, we found the applied flow cytometric methods very useful for the study of chicken T cell biology. PMID:20434546

  15. Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis.

    PubMed

    Henning, Heiko; Petrunkina, Anna M; Harrison, Robin A P; Waberski, Dagmar

    2012-07-01

    The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability. PMID:22573481

  16. FLOW CYTOMETRIC COMPARISON OF THE EFFECTS OF TRIALKYTING ON THE MURINE ERYTHROLEUKEMIC CELL

    EPA Science Inventory

    Cellular effects of exposure to tributyltin (TBT), triethyltin (TET), or trimethyltin (TMT) were investigated by flow cytometry employing the murine erythroleukemic cell (MELC) as a model cellular system. Cell viability was investigated by the carboxyfluorescein diacetate (CFDA) ...

  17. Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis

    PubMed Central

    KONG, FANCONG; ZHANG, LIMING; WANG, HONGXIANG; YUAN, GUOLIN; GUO, ANYUAN; LI, QIUBAI; CHEN, ZHICHAO

    2015-01-01

    Microvesicles (MVs) in body fluids participate in a variety of physical and pathological processes, and are regarded as potential biomarkers for numerous diseases. Flow cytometry (FCM) is among the most frequently used techniques for MV detection. However, different handling methods unavoidably cause pre-analytical variations in the counts and sizes of MVs determined by FCM. The aim of the present study was to investigate the effect of centrifugation, storage conditions and anticoagulant on MV measurements. Blood samples were obtained from 13 healthy donors, including 4 women and 9 men. Calcein-AM staining was used to label MVs and assess the impact of pre-analytical preparation, including centrifugation, and storage conditions on MV measurements obtained using FCM. The range of factors investigated for comparison included: Platelet-free plasma (PFP) stored at −80°C for 1 or 4 weeks; MVs stored at 4°C for 3–4 days or 1 week; MVs frozen at −80°C for 1 or 4 weeks; and anticoagulants, either heparin or ethylenediaminetetraacetic acid (EDTA). No statistically significant differences in MV counts were detected between the two centrifugation speeds (16,000 and 20,500 × g) or among the three centrifugation times (15, 30 and 60 min) investigated. Similarly, no significant differences were noted in MV counts between the two anticoagulants tested (heparin and EDTA). However, the storage of PFP or MVs in heparin-anticoagulated plasma for different periods markedly affected the detected MV counts and size distribution. The counts and sizes of MVs from EDTA-anticoagulated plasma were only affected when the MVs were frozen at −80°C for 4 weeks. In conclusion, calcein-AM is able to efficiently identify MVs from plasma and may be an alternative to Annexin V for MV staining. EDTA preserves the MV counts and size more accurately compared with heparin under calcein-AM staining. PFP centrifuged at 16,000 × g for 15 min is sufficient to isolate MVs, which enables the

  18. Sources of variation in flow cytometric analysis of aquatic species sperm: The effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating.

    PubMed

    Daly, Jonathan; Tiersch, Terrence R

    2012-12-11

    The use of fluorescent staining and flow cytometry to assess sperm quality in aquatic species has increased over the past decade, but comparisons among studies are difficult or impossible due to variation in application, analysis, and reporting of protocols and data.The goal of the present study was to determine the effect of exposure to two cryoprotectants commonly used for cryopreservation of sperm from aquatic species on the accuracy of flow cytometric assessment of sperm quality.Membrane integrity of zebrafish (Danio rerio) sperm exposed to 10% and 20%methanol and dimethyl sulfoxide (DMSO)in 300 mOsm kg(-1) Hanks' balanced salt solution (HBSS) or calcium-free HBSSwas determined using SYBR 14/propidium iodide staining. Both cryoprotectants significantly affected forward-scatter and side-scatter characteristics of sperm samples, resulting in significant changes in the number of total and gated events, and in the number and percentage of intact cells. These results indicate that it cannot be assumed that the approach to flow cytometric analysis of fresh sperm will be applicable to cryoprotectant-treated or cryopreserved sperm. In total, we document examples of five potentially interacting factors that produce errors of 5 to 50% each, resulting in underestimates and overestimates of total and intact sperm (actual numbers and percentages) in the presence of the two most commonly used cryoprotectants at the concentrations used most often for cryopreservation of sperm from aquatic species. This study provides methods to reduce or eliminate these errors and recommendations necessary for standardization and reporting. PMID:23175587

  19. Analysis of synthetic and biological microparticles on several flow cytometric platforms

    EPA Science Inventory

    Microparticles (MPs) are membrane vesicles (0.1 to 1 urn) released from cells upon activation. The limit of detection ofmost standard flow cytometers is just below 1 urn. Recent advances enable detection of particles lower than 0.5 urn, Synthetic. beads are used to define size ra...

  20. FLOW CYTOMETRIC DISCRIMINATION OF MITOTIC NUCLEI BY RIGHT-ANGLE LIGHT SCATTER (JOURNAL VERSION)

    EPA Science Inventory

    Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocynate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 p...

  1. FLOW CYTOMETRIC ANALYSIS OF EFFECTS OF 1,3-DINITROBENZENE ON RAT SPERMATOGENESIS

    EPA Science Inventory

    Exposure of 100-d old rats to 1,3-dinitiobenzene (m-DNB) at dosages up to 48 mg/kg resulted in disruption of spermatogenesis as measured by flow cytometry (FCM) of acridine orange-stained sperm and testis cells. ne day (d 1) after a single exposure to 48 mg/kg m-DNB. CM measureme...

  2. FLOW CYTOMETRIC ANALYSIS OF MOUSE SPERMATOGENIC FUNCTION FOLLOWING EXPOSURE TO ETHYLNITROSOUREA

    EPA Science Inventory

    The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements. Groups of mice received daily exposures ranging from 0 to 75 mg/kg bo...

  3. IMPROVED FLOW CYTOMETRIC ASSAY FOR SOMATIC MUTATIONS AT THE GLYCOPHORIN A LOCUS IN HUMANS

    EPA Science Inventory

    An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. he new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described 1W1 assay that required a...

  4. Flow Cytometric and 16S Sequencing Methodologies for Monitoring the Physiological Status of the Microbiome in Powdered Infant Formula Production

    PubMed Central

    Anvarian, Amir H. P.; Cao, Yu; Srikumar, Shabarinath; Fanning, Séamus; Jordan, Kieran

    2016-01-01

    The aim of this study was to develop appropriate protocols for flow cytometric (FCM) and 16S rDNA sequencing investigation of the microbiome in a powdered infant formula (PIF) production facility. Twenty swabs were collected from each of the three care zones of a PIF production facility and used for preparing composite samples. For FCM studies, the swabs were washed in 200 mL phosphate buffer saline (PBS). The cells were harvested by three-step centrifugation followed by a single stage filtration. Cells were dispersed in fresh PBS and analyzed with a flow cytometer for membrane integrity, metabolic activity, respiratory activity and Gram characteristics of the microbiome using various fluorophores. The samples were also plated on agar plates to determine the number of culturable cells. For 16S rDNA sequencing studies, the cells were harvested by centrifugation only. Genomic DNA was extracted using a chloroform-based method and used for 16S rDNA sequencing studies. Compared to the dry low and high care zones, the wet medium care zone contained a greater number of viable, culturable, and metabolically active cells. Viable but non-culturable cells were also detected in dry-care zones. In total, 243 genera were detected in the facility of which 42 were found in all three care zones. The greatest diversity in the microbiome was observed in low care. The genera present in low, medium and high care were mostly associated with soil, water, and humans, respectively. The most prevalent genera in low, medium and high care were Pseudomonas, Acinetobacter, and Streptococcus, respectively. The integration of FCM and metagenomic data provided further information on the density of different species in the facility. PMID:27446009

  5. Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed.

    PubMed

    Bienenmann-Ploum, Monique E; Vincent, Ursula; Campbell, Katrina; Huet, Anne-Catherine; Haasnoot, Willem; Delahaut, Philippe; Stolker, Linda A A M; Elliott, Christopher T; Nielen, Michel W F

    2013-11-01

    Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D(®)) and a more cost-efficient and transportable planar imaging detector (MAGPIX(®)), hence demonstrating adequate transferability. PMID:24081566

  6. Use of Flow Cytometric Methods to Quantify Protein-Protein Interactions

    PubMed Central

    Blazer, Levi L.; Roman, David L.; Muxlow, Molly R.; Neubig, Richard R.

    2010-01-01

    A method is described for the quantitative analysis of protein-protein interactions using the Flow Cytometry Protein Interaction Assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead-associated fluorescence in a flow cytometer. This method can be used to calculate protein-protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the Regulator of G-Protein Signaling protein, RGS19, in either a saturation or competition format. An adaptation of this method that is compatible for High Throughput screening is also provided. PMID:20069525

  7. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes

    PubMed Central

    van Dongen, J J M; Lhermitte, L; Böttcher, S; Almeida, J; van der Velden, V H J; Flores-Montero, J; Rawstron, A; Asnafi, V; Lécrevisse, Q; Lucio, P; Mejstrikova, E; Szczepański, T; Kalina, T; de Tute, R; Brüggemann, M; Sedek, L; Cullen, M; Langerak, A W; Mendonça, A; Macintyre, E; Martin-Ayuso, M; Hrusak, O; Vidriales, M B; Orfao, A

    2012-01-01

    Most consensus leukemia & lymphoma antibody panels consist of lists of markers based on expert opinions, but they have not been validated. Here we present the validated EuroFlow 8-color antibody panels for immunophenotyping of hematological malignancies. The single-tube screening panels and multi-tube classification panels fit into the EuroFlow diagnostic algorithm with entries defined by clinical and laboratory parameters. The panels were constructed in 2–7 sequential design–evaluation–redesign rounds, using novel Infinicyt software tools for multivariate data analysis. Two groups of markers are combined in each 8-color tube: (i) backbone markers to identify distinct cell populations in a sample, and (ii) markers for characterization of specific cell populations. In multi-tube panels, the backbone markers were optimally placed at the same fluorochrome position in every tube, to provide identical multidimensional localization of the target cell population(s). The characterization markers were positioned according to the diagnostic utility of the combined markers. Each proposed antibody combination was tested against reference databases of normal and malignant cells from healthy subjects and WHO-based disease entities, respectively. The EuroFlow studies resulted in validated and flexible 8-color antibody panels for multidimensional identification and characterization of normal and aberrant cells, optimally suited for immunophenotypic screening and classification of hematological malignancies. PMID:22552007

  8. Flow cytometric evaluation of sperm parameters in relation to fertility potential.

    PubMed

    Gillan, Lindsay; Evans, Gareth; Maxwell, W M C

    2005-01-15

    Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3'-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used

  9. Flow cytometric analysis to detect pathogens in bacterial cell mixtures using semiconductor quantum dots.

    PubMed

    Hahn, Megan A; Keng, Peter C; Krauss, Todd D

    2008-02-01

    Compared to a common green organic dye, semiconductor quantum dots (QDs) composed of CdSe/ZnS core/shell bioconjugates display brighter fluorescence intensities, lower detection thresholds, and better accuracy in analyzing bacterial cell mixtures composed of pathogenic E. coli O157:H7 and harmless E. coli DH5alpha using flow cytometry. For the same given bacterial mixture, QDs display fluorescence intensity levels that are approximately 1 order of magnitude brighter compared to the analogous experiments that utilize the standard dye fluorescein isothiocyanate. Detection limits are lowest when QDs are used as the fluorophore label for the pathogenic E. coli O157:H7 serotype: limits of 1% O157:H7 in 99% DH5alpha result, corresponding to 106 cells/mL, which is comparable to other developing fluorescence-based techniques for pathogen detection. Finally, utilizing QDs to label E. coli O157:H7 in cell mixtures results in greater accuracy and more closely approaches the ideal fluorophore for pathogen detection using flow cytometry. With their broader absorption spectra and narrower emission spectra than organic dyes, QDs can make vast improvements in the field of flow cytometry, where single-source excitation and simultaneous detection of multicolor species without complicating experimental setups or data analysis is quite advantageous for analyzing heterogeneous cell mixtures, both for prokaryotic pathogen detection and for studies on eukaryotic cell characteristics. PMID:18186615

  10. Flow cytometric detection of spontaneous apoptosis in human breast cancer using the TUNEL-technique.

    PubMed

    Ehemann, Volker; Sykora, Jaromir; Vera-Delgado, Jorge; Lange, Adelheid; Otto, Herwart F

    2003-05-01

    Microscopic detection of structural alterations is the most reliable method to identify apoptotic cells, which however, does not allow any correlation with cell cycle phases. Discrimination of individual cells within solid human tumors undergoing apoptotic death is possible by flow cytometry where apoptotic cells appear in a hypodiploid sub G0/1-peak as a consequence of partial DNA loss. To refer induction of apoptosis to cell cycle phases we adopted the terminal deoxynucleotidyl transferase nick-end-labelling (TUNEL) technique to flow cytometry which enables the detection of cellular DNA content and DNA fragmentation by multiparametric analysis. One thousand seven hundred human breast carcinomas were screened. In 40 cases (2.3%) of 1700 carcinomas we detected a hypodiploid sub -G0/1 apoptotic peak. The spontaneous apoptotic fractions within individual tumors ranged between 1.5 and 25%. A correlation (r(2)=0.78) was found between apoptotic cells in sub-G0/1-peak measured by DNA-cytometry and TUNEL positive cells measured by multiparametric cytometry, because TUNEL reaction signed also cells with strand breaks. High proliferation indices correspond well (r(2)=0.807) with the increased amount of TUNEL positive cells. Multiparametric flow cytometry for the combined determination of DNA-content and DNA-fragmentation by TUNEL offers not only the advantage of a higher apoptosis sensitivity but also enables the quantification of DNA fragmentation related to any cell cycle phase. PMID:12706866

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

    NASA Astrophysics Data System (ADS)

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

    2005-04-01

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

  12. Detection and quantification of circulating immature platelets: agreement between flow cytometric and automated detection.

    PubMed

    Ibrahim, Homam; Nadipalli, Srinivas; Usmani, Saba; DeLao, Timothy; Green, LaShawna; Kleiman, Neal S

    2016-07-01

    Immature platelets-also termed reticulated platelets (RP)-are platelets newly released into the circulation, and have been associated with a variety of pathological thrombotic events. They can be assessed by flow cytometry after staining with thiazole orange (TO) or by using a module added to a fully automated analyzer that is currently in wide clinical use and expressed as a fraction of the total platelet count (IPF). We sought to assess the correlation and agreement between these two methods. IPF was measured using Sysmex XE 2100-and at the same time point- we used TO staining and flow cytometry to measure RP levels. Two different gates were used for the flow cytometry method, 1 and 0.5 %. Measurements from the automated analyzer were then compared separately to measurements performed using each gate. Agreement between methods was assessed using Bland-Altman method. Pearson's correlation coefficient was also calculated. 129 subjects were enrolled and stratified into 5 groups: (1) Healthy subjects, (2) End stage renal disease, (3) Chronic stable coronary artery disease, (4) Post Coronary artery bypass surgery, (5) Peripheral thrombocytopenia. Median IPF levels were increased for patients in groups 2, 3, 4 and 5 (4.0, 4.7, 4.3, and 8.3 % respectively) compared to healthy subjects (2.5 %) p = 0.0001. Although the observed correlation between the two methods tended to be good in patients with high IPF values (i.e., group 5), the overall observed correlation was poor (Pearson's correlation coefficient r = 0.27). Furthermore, there was poor agreement between the two methods in all groups. Despite the good correlation that was observed between the two methods at higher IPF values, the lack of agreement was significant. PMID:26831482

  13. Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms.

    PubMed

    Davey, H M; Kell, D B

    1997-08-01

    Flow cytometry is a rapid method for measuring the optical properties of individual cells. The technique has found great utility in the study of mammalian cells, but microbiological applications have been more limited. We here show that UV-excited fluorescent whitening agents, in particular Tinopal CBS-X, are effective stains for both vegetative microbial cells and for spores of Gram-positive bacteria. Pretreatment of samples with ethanol speeds the staining process. Under favourable conditions, Tinopal CBS-X may be used to discriminate among organisms, a fact that may be useful when screening for a target microorganism against a high biological background. PMID:9266751

  14. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA-Binding and Cleavage Properties

    PubMed Central

    Baxter, Sarah K.; Lambert, Abigail R.; Scharenberg, Andrew M.; Jarjour, Jordan

    2014-01-01

    A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry. PMID:23423888

  15. Discriminating Active Tuberculosis from Latent Tuberculosis Infection by flow cytometric measurement of CD161-expressing T cells

    PubMed Central

    Yang, Qianting; Xu, Qian; Chen, Qi; Li, Jin; Zhang, Mingxia; Cai, Yi; Liu, Haiying; Zhou, Yiping; Deng, Guofang; Deng, Qunyi; Zhou, Boping; Kornfeld, Hardy; Chen, Xinchun

    2015-01-01

    Interferon-gamma Release Assays (IGRAs) significantly increases the possibility for early diagnosis of tuberculosis, but IGRAs alone cannot discriminate active TB from LTBI. Therefore, fast and reliable discrimination of active tuberculosis, especially bacteriology negative tuberculosis, from LTBI is a great necessity. Here we established an assay based on flow cytometric multiparameter assay assessing expression of CD161 along with CD3, CD4, and CD8, whereby a set of indices formulated by the percentages of CD3+CD161+, CD3+CD4+CD161+ and CD3+CD8+CD161+ T cells multiplied with lymphocyte/monocyte ratio were established. Application of the CD3+CD8+CD161+ index to compare a cohort of active tuberculosis with a cohort of LTBI or health control yielded 0.7662 (95% confidence interval [CI] 0.6559–0.8552) or 0.7922 (95%  CI 0.6846–0.8763) for sensitivity and 0.9048 (95%  CI 0.8209–0.9580) or 0.8939 (95% CI 0.8392–0.9349) for specificity when the TB cohort was AFB+; the corresponding results were 0.7481 (95%  CI 0.6648–0.8198) or 0.7557 (95%  CI 0.6730–0.8265) for sensitivity and 0.8571 (95%  CI 0.7637–0.9239) or 0.8603 (95%  CI 0.8008–0.9075) for specificity when the TB cohort was AFB−. Our results reveal that in combination with IGRAs, CD161-based indices provide a novel, fast diagnostic solution addressing the limitation of current tuberculosis diagnostics. PMID:26643453

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

    PubMed

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

    2016-07-01

    Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. Here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. However, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). By just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. Furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. Taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system. PMID:27142297

  17. Analysis of informativeness of immunohistochemical and flow cytometric methods for estrogen receptor α assessment.

    PubMed

    Bogush, T A; Dudko, E A; Rodionova, M V; Bogush, E A; Kirsanov, V J; Rodionov, V V; Vorotnikov, I K

    2015-01-01

    Informative capacity analysis of immunohistochemistry (IHC) and flow cytometry (FCM) in the assessment of estrogen receptor α (ERα) expression in breast cancer tissue was performed. Similar frequencies of expression were shown by both methods: 27% of ERα-negative and 73% ERα-positive cases. However, IHC evaluation detected low levels in only 20% of ERα-positive cases, whereas low levels of ERα detected by FCM were 2 times more often (48%). Moreover, FCM revealed positive expression (23-60%) in 33% of IHC ERα-negative cases. Among IHC ER-positive cases, zero ERα expression was detected by FCM in 12.5%. The approaches to minimize errors in routine clinical determination of the estrogen receptor status were proposed. PMID:26728725

  18. Flow cytometric monitoring of hormone receptor expression in human solid tumors

    NASA Astrophysics Data System (ADS)

    Krishan, Awtar

    2002-05-01

    Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

  19. High speed flow cytometric detection of rare glycophorin A mutations in human blood cells

    SciTech Connect

    Langlois, R.G.; Engh, G. van den )

    1993-01-01

    The glycophorin A (GPA) assay utilizes immunofluorescent labeling and flow cytometry to measure the frequency of peripheral erythrocytes with mutant phenotypes, presumably due to mutations in erythroid precursor cells. Analysis of 5 [times] 10[sup 6] cells/assay is used to enumerate variant erythrocytes that occur at a frequency of 3-10 [times] 10[sup [minus]6] in unexposed donors. Extension of this assay to human reticulocytes requires detection of variants that occur at frequencies as low as 3 [times] 10[sup [minus]8]. The authors have used high speed data acquisition and cell classification electronics to perform 3-color analysis at rates up to 20,000 cells/s. High speed analysis of up to 10[sup 8] cells/assay has been used to enumerate GPA-variant reticulocytes in normal donors.

  20. Flow cytometric determination of intracellular or secreted IFNgamma for the quantification of antigen reactive T cells.

    PubMed

    Asemissen, A M; Nagorsen, D; Keilholz, U; Letsch, A; Schmittel, A; Thiel, E; Scheibenbogen, C

    2001-05-01

    The detection of antigen-induced IFNgamma secretion at the single cell level can be used to identify and enumerate antigen-reactive T cells from peripheral blood. This study was performed to analyze the suitability of T cell enumeration by flow cytometry in comparison with the ELISPOT assay. Peripheral blood mononuclear cell (PBMC) samples from six HLA-A2+ healthy subjects were analysed for the frequency of influenza-reactive CD8+ T cells by flow cytometry detecting either intracellular IFNgamma (IC-FC) or secreted IFNgamma (S-FC). All samples were also analysed by IFNgamma ELISPOT assay. The frequency of influenza peptide-reactive T cells determined by IC-FC was 0.01 to 0.34% of CD8+ T cells and by ELISPOT assay 0.02 to 0.23% of CD8+ T cells (n=6 subjects) with a high inter-assay reproducibility and a close correlation between the assays (r=0.77, P<0.001). Little or no IFNgamma production was observed in unstimulated PBMC samples using either the IC-FC or the ELISPOT assay. In contrast, using S-FC large numbers of IFNgamma-secreting CD8+ T cells (0.37% to 5.55%, n=6 subjects) were detected in unstimulated PBMC. The frequency of influenza-reactive CD8+ T cells (0.57-5.19%, n=6 subjects) determined by S-FC did not correlate with the values from the IC-FC or ELISPOT assays. This comparative study shows the suitability of the determination of frequencies of antigen reactive T cells in PBMC by IC-FC. The advantage of IC-FC is the possibility to phenotype simultaneously antigen-reactive T cells. PMID:11292486

  1. Ciliate ingestion and digestion: flow cytometric measurements and regrowth of a digestion-resistant campylobacter jejuni

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a method to measure ingestion and digestion rates of bacterivorous protists feeding on pathogenic bacteria. We tested this method using the enteric bacteria Campylobacter jejuni and a freshwater colpodid ciliate. Campylobacter and a non-pathogenic bacteria isolated from the environment ...

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

    USGS Publications Warehouse

    Jenkins, Jill A.

    2013-01-01

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

  3. Complex karyotypes in flow cytometrically DNA-diploid squamous cell carcinomas of the head and neck.

    PubMed Central

    Akervall, J.; Jin, Y.; Baldetorp, B.; Mertens, F.; Wennerberg, J.

    1998-01-01

    In squamous cell carcinoma of the head and neck (SCCHN), DNA ploidy as determined by flow cytometry (FCM) has been found to yield prognostic information but only for tumours at oral sites. Cytogenetic findings have indicated complex karyotype to be a correlate of poor clinical outcome. In the present study, 73 SCCHN were investigated with the two techniques. Aneuploid cell populations were identified in 49 (67%) cases by FCM but in only 21 (29%) cases by cytogenetic analysis. The chromosome index (CI), calculated as the mean chromosome number divided by 46, was compared with the respective DNA index (DI) obtained by FCM in 15 tumours, non-diploid according to both techniques, DI being systematically 12% higher than CI in this subgroup. Eight (33%) of the 24 tumours diploid according to FCM had complex karyotypes, three of the tumours being cytogenetically hypodiploid, three diploid and two non-diploid. The findings in the present study may partly explain the low prognostic value of ploidy status as assessed by FCM that has been observed in SCCHN. In addition, we conclude that FCM yields information of the genetic changes that is too unspecific, and that cytogenetic analysis shows a high rate of unsuccessful investigations, thus diminishing the value of the two methods as prognostic factors in SCCHN. Images Figure 1 PMID:9569043

  4. Comparison of quantitative flow cytometric data provided by panels with lower and increased color number

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Mittag, Anja; Pierzchalski, Arkadiusz; Baumgartner, Adolf; Dähnert, Ingo; Tárnok, Attila

    2012-03-01

    To date the flow cytometry (FCM) industry is booming with new generations of commercial clinical instruments. Long-term clinical studies have the dilemma that moving to new instruments being capable of more complex cell-analysis makes it difficult to compare new data with those obtained on older instruments with less complex analysis panels. Since 15 years we conduct follow-up studies on children with congenital heart diseases. In this period we moved from 2- to 3- and now to 10-color FCM immunophenotyping panels. Questions arise how to compare and transfer data from lower to higher level of complexity. Two comparable antibody panels for leukocyte immunophenotyping (12-tube 2-colors, and 9-tube 4-colors) were measured on a BD FACScalibur FCM (calibration: Spherotech beads) in 19 blood samples from children with congenital heart disease. This increase of colors was accompanied by moving antibodies that were in the 2-color panel either FITC or PE labeled to red dyes such as PerCP or APC. Algorithms were developed for bridging data for quantitative characterization of antigen expression (mean fluorescence intensity) and frequency of different cell subpopulations in combination with rainbow bead standard data. This approach worked for the most relevant antibodies (CD3, CD4, CD8 etc.) well, but rendered substantial uncertainty for activation markers (CD69 etc.). Our techniques are particularly well suited to the analysis in long-term studies and have the potential to compare older and recent results in a standardized way.

  5. Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

    PubMed Central

    Boltze, Johannes; Wagner, Daniel-Christoph; Weise, Gesa

    2016-01-01

    Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed. Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair. PMID:26967380

  6. Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution.

    PubMed

    Dalgaard, T S; Norup, L R; Rubbenstroth, D; Wattrang, E; Juul-Madsen, H R

    2010-11-15

    Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αβ-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future. PMID:20739071

  7. Weekly flow cytometric analysis of riverine phytoplankton to determine seasonal bloom dynamics.

    PubMed

    Read, Daniel S; Bowes, Michael J; Newbold, Lindsay K; Whiteley, Andrew S

    2014-03-01

    Understanding the relative role of anthropogenic and environmental drivers on the timing, magnitude and composition of algal and cyanobacterial blooms is vitally important for the effective management of river catchments. Whilst taxonomic identification and enumeration of algal species can provide valuable insights, the time and specialist skills needed for this approach makes it prohibitive for high frequency and multiple-site studies. Other proxies for phytoplankton, such as total chlorophyll concentration provide little information on community composition. Here we demonstrate the use of flow cytometry (FCM) as a viable alternative approach for monitoring the changing seasonal patterns of abundance, composition and biovolume of phytoplankton in rivers. A FCM assay was set up and calibrated using a range of pure algal cultures and then applied to a year-long, weekly sampling campaign on the River Thames at Wallingford, UK. Ten groups of phytoplankton representing diatoms, chlorophytes, cryptophytes and cyanobacteria were monitored over the course of the year and examined in relation to river physiochemical parameters. Major diatom blooms occurred in spring and autumn, correlating with depletion of soluble reactive phosphorus and dissolved silicon concentrations and we also observed a significant and sustained cyanobacteria bloom between July and October. Pico-chlorophytes (0.2-2.0 μm in diameter) dominated the community throughout the summer period but were not detected using traditional colorimetric chlorophyll analysis, suggesting underestimates of actual phytoplankton standing stocks by traditional methods. We demonstrate high resolution sampling and FCM as a sensitive method for river ecosystem monitoring and that FCM data may be used as an indicator of riverine health. PMID:24510006

  8. DNA fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm.

    PubMed

    Kjelland, M E; González-Marín, C; Gosálvez, J; López-Fernández, C; Lenz, R W; Evans, K M; Moreno, J F

    2011-12-01

    This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials). PMID:21788426

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

    PubMed

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

    2006-06-01

    The effectiveness of solar disinfection (SODIS), a low-cost household water treatment method for developing countries, was investigated with flow cytometry and viability stains for the enteric bacterium Escherichia coli. A better understanding of the process of injury or death of E. coli during SODIS could be gained by investigating six different cellular functions, namely: efflux pump activity (Syto 9 plus ethidium bromide), membrane potential [bis-(1,3-dibutylbarbituric acid)trimethine oxonol; DiBAC4(3)], membrane integrity (LIVE/DEAD BacLight), glucose uptake activity (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose; 2-NBDG), total ATP concentration (BacTiter-Glo) and culturability (pour-plate method). These variables were measured in E. coli K-12 MG1655 cells that were exposed to either sunlight or artificial UVA light. The inactivation pattern of cellular functions was very similar for both light sources. A UVA light dose (fluence) of <500 kJ m(-2) was enough to lower the proton motive force, such that efflux pump activity and ATP synthesis decreased significantly. The loss of membrane potential, glucose uptake activity and culturability of >80 % of the cells was observed at a fluence of approximately 1500 kJ m(-2), and the cytoplasmic membrane of bacterial cells became permeable at a fluence of >2500 kJ m(-2). Culturable counts of stressed bacteria after anaerobic incubation on sodium pyruvate-supplemented tryptic soy agar closely correlated with the loss of membrane potential. The results strongly suggest that cells exposed to >1500 kJ m(-2) solar UVA (corresponding to 530 W m(-2) global sunlight intensity for 6 h) were no longer able to repair the damage and recover. Our study confirms the lethal effect of SODIS with cultivation-independent methods and gives a detailed picture of the 'agony' of E. coli when it is stressed with sunlight. PMID:16735735

  10. Modulation of TGF-beta type 1 receptor: flow cytometric detection with biotinylated TGF-beta.

    PubMed

    Newman, W; Beall, L D; Bertolini, D R; Cone, J L

    1989-10-01

    Transforming growth factor beta type 1 (TGF-beta 1) was reacted with NHS-biotin to yield a derivative of TGF-beta 1 which was biotinylated on lysine residues. The biotinylated form of TGF-beta 1 was separated from the unreacted material by reverse phase chromatography. In three separate bioassays, the derivatized peptide was as active as the starting material. The use of FITC-avidin in conjunction with flow cytometry demonstrated that the binding of biotinylated TGF-beta 1 to its receptor is saturable, competable, and specific. A 100-fold molar excess of underivatized TGF-beta 1 gave 85% inhibition of binding of the biotinylated peptide to the mink lung cell line CCL-64, while TGF-beta 2 showed no inhibition of binding, nor did insulin, calcitonin, or TGF-alpha. Both CCL-64 cells and human umbilical vein endothelial cells showed a density-dependent down-regulation of receptor expression in culture. Several factors were examined that might mediate this effect. The down-regulation was shown not to be due to the secretion of an active form of TGF-beta 1. The extracellular matrix from high-density cells did not decrease expression of the receptor. Fibronectin, collagen, and gelatin were also unable to signal changes in receptor expression, even though in other systems such matrix components can regulate the responsiveness of cells to TGF-beta 1. Lastly, staining simultaneously for DNA content and TGF-beta 1 receptor expression showed that there was no correlation between cell cycle and receptor levels. PMID:2550480

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

    PubMed

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

    1995-09-01

    Using four cell lines including drug-sensitive K562/Parent cells, P-glycoprotein (Pgp)-mediated multidrug resistant (MDR) K562/VCR, K562/ADR and revertant K562/ADR-R cells, two fluorescent agents, Fluo-3 and rhodamine-123 (Rh-123), were compared as indicators in a functional assay of MDR. Cells were incubated with 4 microM Fluo-3 or 1 microM Rh-123 for 45 min and then the intracellular accumulation of the agent was measured using a flow cytometer. Verapamil (20 microM) or cepharanthine (biscoclaurine alkaloid, 10 microM) was added just before the fluorescent agents. Efflux patterns were also studied 60 min after incubation with or without verapamil and cepharanthine. Increased intracellular accumulation and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were demonstrated in multidrug resistant K562/VCR and K562/ADR cells, indicating that Fluo-3 is another good indicator of MDR. However, a similar, but lower, increase in uptake and a delayed efflux pattern of Fluo-3 by verapamil and cepharanthine were also demonstrated even in Pgp-non-overexpressed K562/Parent cells. In contrast, accumulation of Rh-123 was not affected by verapamil and cepharanthine. To further study the Pgp dependency of Fluo-3, another cell line, K562/NC16 expressing minimum MDR1 mRNA, was cloned. Increased uptake and a delayed efflux pattern of Fluo-3, but not Rh-123, with verapamil or cepharanthine were again demonstrated in K562/NC16 cells, indicating that intracellular accumulation of Fluo-3 may be non-specifically influenced by verapamil and cepharanthine at very low levels of Pgp-related MDR, while the influx and efflux patterns of Rh-123 may be specifically affected by Pgp overexpression. PMID:7488425

  12. Flow cytometric determination of genome size for eight commercially important fish species in China.

    PubMed

    Zhu, Dongmei; Song, Wen; Yang, Kun; Cao, Xiaojuan; Gul, Yasmeen; Wang, Weiming

    2012-09-01

    The genome size (C value) of eight commercially important fish species in China was measured using flow cytometry. Chicken (Gallus domesticus) erythrocytes were used as reference cells. When using propidium iodide (PI) as the fluorescent dye, genome sizes were 1.09 ± 0.08, 2.75 ± 0.12, 1.05 ± 0.05, 1.35 ± 0.11, 0.99 ± 0.05, 0.90 ± 0.08, 0.90 ± 0.07, and 0.88 ± 0.07 pg for Japanese eel (Anguilla japonica), mullet (Myxocyprinus asiaticus), yellowcheek carp (Elopichthys bambusa), blunt snout bream (Megalobrama amblycephala), yellow catfish (Pelteobagrus fulvidraco), ricefield eel (Monopterus albus), mandarin fish (Siniperca chuatsi), and snakehead (Ophicephalus argus), respectively. However, genome sizes were 1.25 ± 0.00, 3.08 ± 0.02, 1.25 ± 0.00, 1.57 ± 0.01, 0.96 ± 0.01, 1.00 ± 0.01, 0.91 ± 0.01, and 0.89 ± 0.01 pg for these fishes, respectively, when 4', 6-diamidino-2-phenylindole (DAPI) was used as the fluorescent dye. Regardless of the dye used, the more evolutionarily advanced species had a smaller genome size than those with a lower evolutionary status. For each species, we also measured the size of erythrocytes and their nucleus and evaluated the relationships between erythrocyte size, nucleus size, chromosome number, and genome size. Genome size was positively correlated with erythrocyte nucleus size and chromosome number when using PI as the fluorescent dye, but it was only correlated with erythrocyte nucleus size when DAPI was used. PMID:22956044

  13. Image and flow cytometric analysis of gold nanoparticle uptake by macrophages

    NASA Astrophysics Data System (ADS)

    Fixler, Dror; Ankri, Rinat; Weiss, Ronald; Grahnert, Anja; Melzer, Susanne; Tárnok, Attila

    2016-03-01

    Background/Aim: In atherosclerosis stable and vulnerable atherosclerotic plaque types are distinguished that behave differently concerning rupture, thrombosis and clinical events. The stable are rich in M2 macrophages. The unstable are rich in inflammatory M1 macrophages and are highly susceptible to rupture, setting patients at risk for thrombotic events when they undergo invasive diagnosis such as coronary angiography. Therefore, novel approaches for non-invasive detection and classification of vulnerable plaques in vivo are needed. Whereas classical approaches fail to differentiate between both plaque types, a new biophotonic method (combination of the diffusion reflection (DR) method with flow cytometry (FCM) or image cytometry (IC)) to analyze gold nanoparticle (GNP) loading of plaques could overcome this limitation. Methods: Two types of GNP were used three variants of gold nanorods (GNRI with 40x18 nm, II 65x25 nm and III 52x13 nm in size) and gold nanospheres (GNS with an average diameter of 18.5 nm). The GNS had an absorption peak at 520 nm and the GNR at 630 nm. Monocytes were isolated from human buffy blood samples, differentiated into macrophages and their subtypes and labelled with GNR and GNS for 3 and 24 h. GNS and GNR loading were determined by FCM and/or IC. Macrophages within tissue-like phantoms were analyzed by the DR system. Results: After GNR labelling of macrophages the FCM light scatter values increased up to 3.7 fold and the DR slope changed from an average slope of 0.196 (macrophages only) to an average slope of 0.827 (macrophages labelled with GNR). But, GNRIII did not present much higher DR slopes than the control phantoms, indicating that macrophages take up GNRIII in a lower amount than GNRI or II. IC and microscopy showed that all particle variants were taken up by the cells in a heterogeneous fashion. Conclusion and outlook: The combination of FCM and DR measurements provides a potential novel, highly sensitive and non

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

    USGS Publications Warehouse

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

    1998-01-01

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

  15. 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.

    PubMed

    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. PMID:17803189

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

    PubMed

    Rose, Jonathan A; Wanner, Nicholas; Cheong, Hoi I; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

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

    PubMed Central

    Rose, Jonathan A.; Wanner, Nicholas; Cheong, Hoi I.; Queisser, Kimberly; Barrett, Patrick; Park, Margaret; Hite, Corrine; Naga Prasad, Sathyamangla V.; Erzurum, Serpil; Asosingh, Kewal

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a heterogeneous disease characterized by severe angiogenic remodeling of the pulmonary artery wall and right ventricular hypertrophy. Thus, there is an increasing need for novel biomarkers to dissect disease heterogeneity, and predict treatment response. Although β-adrenergic receptor (βAR) dysfunction is well documented in left heart disease while endothelial cell-derived microparticles (Ec-MPs) are established biomarkers of angiogenic remodeling, methods for easy large clinical cohort analysis of these biomarkers are currently absent. Here we describe flow cytometric methods for quantification of βAR density on circulating white blood cells (WBC) and Ec-MPs in urine samples that can be used as potential biomarkers of right heart failure in PAH. Biotinylated β-blocker alprenolol was synthesized and validated as a βAR specific probe that was combined with immunophenotyping to quantify βAR density in circulating WBC subsets. Ec-MPs obtained from urine samples were stained for annexin-V and CD144, and analyzed by a micro flow cytometer. Flow cytometric detection of alprenolol showed that βAR density was decreased in most WBC subsets in PAH samples compared to healthy controls. Ec-MPs in urine was increased in PAH compared to controls. Furthermore, there was a direct correlation between Ec-MPs and Tricuspid annular plane systolic excursion (TAPSE) in PAH patients. Therefore, flow cytometric quantification of peripheral blood cell βAR density and urinary Ec-MPs may be useful as potential biomarkers of right ventricular function in PAH. PMID:27270458

  18. Flow cytometric analysis of the stimulatory response of T cell subsets from normal and HIV-1+ individuals to various mitogenic stimuli in vitro.

    PubMed Central

    Medina, E; Borthwick, N; Johnson, M A; Miller, S; Bofill, M

    1994-01-01

    A novel technique is described which allows the study of the responses of T cell subpopulations stimulated in bulk cultures without interfering with cell-cell interactions. The number and phenotype of lymphoblasts developing following stimulation with phytohaemagglutinin (PHA), anti-CD3, staphylococcal protein A (SPA) and pokeweed mitogen (PWM) was determined in HIV-1- and HIV-1+ patients using a new five-parameter flow cytometric method. We found that normal T cells responded faster to PHA than to any of the other mitogens tested. The peak of the PHA response occurred on day 3, followed by anti-CD3 and SPA on day 4 and PWM mitogen on day 5. Although PHA and anti-CD3 stimulated up to 95% and 80% of lymphocytes, respectively, SPA and PWM stimulated only 40% and 30% of cells, respectively. A defective T cell response was observed in lymphocytes cultured from asymptomatic HIV-1+ patients compared with negative controls. This loss of response was related to a selective mortality of T cells following mitogenic stimulation, referred to as activation-associated lymphocyte death (AALD). The results showed that stronger mitogens (PHA and anti-CD3) induced AALD in a larger proportion (50-60%) of T cells than weaker mitogens such as SPA and PWM (30-40%), and that AALD affected different lymphocyte subsets to different extents. AALD occurred more frequently in total CD8+ and CD45RO+ T cells compared with CD4+ and CD45RA+ T cells, but memory CD4+ T cells were the population most severely affected in samples from HIV-1+ donors. PMID:7914156

  19. Comparison of methodological data measurement limits in CD4⁺ T lymphocyte flow cytometric enumeration and their clinical impact on HIV management.

    PubMed

    Whitby, Liam; Whitby, Alison; Fletcher, Matthew; Helbert, Matthew; Reilly, John T; Barnett, David

    2013-01-01

    UK NEQAS for Leucocyte Immunophenotyping, an ILAC G13:2000 accredited External Quality Assessment (EQA) organization, with over 3000 international laboratories participating in 14 programmes, issues 2 proficiency testing samples of stabilized whole blood to 824 participants in the Immune Monitoring (lymphocyte subset) programme every two months. We have undertaken a study of 58,626 flow cytometric absolute CD4⁺ T lymphocyte count data sets from these laboratories over a 12-year-period (2001-2012) to determine counting method variation in data measurement limits and how this could influence the clinical management of HIV patients. Comparison of relative error and 99.9% confidence limits for absolute CD4⁺ T lymphocyte values was undertaken using dual platform (DP) and single platform (SP) data and showed that the SP consistently outperformed DP, giving lower relative errors and confidence limits at clinically significant absolute CD4⁺ T lymphocyte counts. Our data shows that absolute CD4⁺ T lymphocyte counts should be obtained using single platform technology to reduce the variability at clinically relevant levels. On data where results (irrespective of platform) were below the international treatment threshold of 350 cells/μl, there was no significant misclassification between either SP or DP techniques meaning most patients would receive the correct treatment at the correct time. However, results that were above the treatment level of 350 cells/μl had a significant difference (P = 0.04) between DP and SP platforms, suggesting patients monitored using DP technology were 20% more likely to start therapy prematurely than those monitored with SP technology. PMID:23788473

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

    PubMed

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

    2016-04-01

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

  1. Comparative flow cytometric analysis of DNA-bound PCNA and DNA content as estimators of S-phase cells in cell cultures.

    PubMed

    Bustamante, A S; Guervós, M A; de los Toyos, J R; Dolbeare, F; Sampedro, A

    1996-07-01

    Flow cytometric estimations of S-phase cells were carried out on cultures from three different cell lines and in frozen aliquots. A PCNA-extraction protocol was applied. Measurements of the S fraction estimated from bivariate PCNA/DNA analysis after detergent extraction of DNA non-bound PCNA were compared with those obtained from total DNA histograms (Vindelöv and Christensen's technique, methanol-fixed whole cells and PCNA-extracted nuclei). No significant differences between methods, or between fresh and frozen specimens, were found in the measurements of the percentage of S-phase cells. Nevertheless, nuclei yield following PCNA extraction was highly variable, ranging from 63% to 10% (mean: 26%). In some cases, the extraction was not complete and samples had to be discarded. Usually, boundaries between S-phase events and G0/G1 or G2/M subpopulations were not clearly defined. Because of these shortcomings, and the fact that is more costly and time consuming, the estimation of the S-phase fraction by means of bivariate DNA-bound PCNA/total DNA flow cytometric studies does not seem to surpass that obtained from standard DNA cell cycle analyses. PMID:8844110

  2. Quantitative DNA analysis of fresh solid tumors by flow and image cytometric methods: a comparison using the Roche Pathology Workstation Image Analyzer.

    PubMed

    Ellison, D A; Maygarden, S J; Novotny, D B

    1995-04-01

    The clinical utility of DNA ploidy and cell cycle parameters as prognostic indicators has been demonstrated for selected malignant tumors. Previous quantitative DNA analysis studies have used various tumor sample preparation methods and analyzers. We undertook a pilot study to compare the results of DNA analysis of fresh solid tumors by flow cytometry with the new Roche Pathology Workstation Image Analyzer. Flow cytometric DNA analysis was done on cell suspensions of fine needle aspirates from fresh tumor specimens and analyzed for ploidy and cell cycle statistics with a Becton-Dickinson FACScan Analyzer, using a rectangular model. Small aliquots from these same aspirates were prepared as direct cytologic smears and Feulgen stained for DNA analysis with the Roche Image Analyzer. Additional smears were stained with Diff-Quik for morphologic correlation with DNA histograms. The study group consisted of 40 malignant neoplasms. There was a high correlation between the flow and image DNA indices (R = 0.93, slope = 1.0036, P < 0.001) but a weaker relationship between the flow and image estimated S-phase fractions (R = 0.57, slope = 0.5401, P < 0.01). DNA ploidy categorization for the two methods was concordant in 30 (75%) cases, discordant in seven (17.5%) cases, and equivocal in three (7.5%) cases. In our experience, quantitative DNA analysis of fresh tumor aspirates by flow and image cytometric methods yielded comparable and/or complementary results, with each method having certain advantages and disadvantages. Proposed reasons for false and true discordances and an approach for evaluation are discussed. PMID:7617654

  3. Flow cytometric measurement of the clearance rate in the blue mussel Mytilus edulis and the development of a new individual exposure system for aquatic immunotoxicological studies.

    PubMed

    Duchemin, Matthieu B; Wessel, Nathalie; Fournier, Michel; Auffret, Michel

    2008-05-01

    Animals in poor health condition are not relevant biological models. The current study focused on the use of the clearance rate of Mytilus edulis to assess the gross physiological condition of individuals maintained in stressful experimental conditions. This approach was developed in a new, highly controlled experimental exposure device designed to investigate individual responses in aquatic ecotoxicological studies. Both clearance rate values and immune parameters analysis indicated that the health condition of mussels kept in 50ml tubes for 24h or 48h was not altered compared to controls, while most parameters were depressed after 72h. Moreover, this study confirms the relevance of flow cytometric for the measurement of clearance rate compared to techniques utilizing microscopy. Current results prompted us to perform further 24h chemical exposure using this "in tubo" device. PMID:17905494

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

    USGS Publications Warehouse

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

    2000-01-01

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

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

    PubMed

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

    2016-09-01

    In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells. PMID:27406324

  6. Flow Cytometric Immunophenotyping Is Sensitive for the Early Diagnosis of De Novo Aggressive Natural Killer Cell Leukemia (ANKL): A Multicenter Retrospective Analysis.

    PubMed

    Li, Yi; Wei, Jia; Mao, Xia; Gao, Qingping; Liu, Longlong; Cheng, Ping; Liu, Limei; Zhang, Xinhua; Zhang, Ke; Wang, Jin; Zhu, Li; Zhou, Jianfeng; Zhang, Yicheng; Meng, Li; Sun, Hanying; Li, Dengju; Huang, Mei; Huang, Wei; Deng, Jinniu; Zhang, Donghua

    2016-01-01

    Aggressive natural killer cell leukemia (ANKL) is a fatal hematological neoplasm characterized by a fulminating clinical course and extremely high mortality. Current diagnosis of this disease is not effective during the early stages and it is easily misdiagnosed as other NK cell disorders. We retrospectively analyzed the clinical characteristics and flow cytometric immunophenotype of 47 patients with ANKL. Patients with extranodal NK/T cell lymphoma, nasal type (ENKTL) and chronic lymphoproliferative disorder of NK cell (CLPD-NK), who were diagnosed during the same time period were used for comparisons. Abnormal NK cells in ANKL were found to have a distinctiveCD56bright/CD16dim immunophenotype and markedly increased Ki-67 expression, whereas CD57 negativity and reduced expression of killer immunoglobulin-like receptor (KIR), CD161, CD7, CD8 and perforin were exhibited compared with other NK cell proliferative disorders (p<0.05). The positive rates of flow cytometry detection (97.4%) was higher than those of cytomorphological (89.5%), immunohistochemical (90%), cytogenetic (56.5%) and F-18 fluorodeoxyglucose positron emission tomography/computer tomography (18-FDG-PET/CT) examinations (50%) (p<0.05). ANKL is a highly aggressive leukemia with high mortality. Flow cytometry detection is sensitive for the early and differential diagnosis of ANKL with high specificity. PMID:27483437

  7. Flow Cytometric Immunophenotyping Is Sensitive for the Early Diagnosis of De Novo Aggressive Natural Killer Cell Leukemia (ANKL): A Multicenter Retrospective Analysis

    PubMed Central

    Mao, Xia; Gao, Qingping; Liu, Longlong; Cheng, Ping; Liu, Limei; Zhang, Xinhua; Zhang, Ke; Wang, Jin; Zhu, Li; Zhou, Jianfeng; Zhang, Yicheng; Meng, Li; Sun, Hanying; Li, Dengju; Huang, Mei; Huang, Wei; Deng, Jinniu; Zhang, Donghua

    2016-01-01

    Aggressive natural killer cell leukemia (ANKL) is a fatal hematological neoplasm characterized by a fulminating clinical course and extremely high mortality. Current diagnosis of this disease is not effective during the early stages and it is easily misdiagnosed as other NK cell disorders. We retrospectively analyzed the clinical characteristics and flow cytometric immunophenotype of 47 patients with ANKL. Patients with extranodal NK/T cell lymphoma, nasal type (ENKTL) and chronic lymphoproliferative disorder of NK cell (CLPD-NK), who were diagnosed during the same time period were used for comparisons. Abnormal NK cells in ANKL were found to have a distinctiveCD56bright/CD16dim immunophenotype and markedly increased Ki-67 expression, whereas CD57 negativity and reduced expression of killer immunoglobulin-like receptor (KIR), CD161, CD7, CD8 and perforin were exhibited compared with other NK cell proliferative disorders (p<0.05). The positive rates of flow cytometry detection (97.4%) was higher than those of cytomorphological (89.5%), immunohistochemical (90%), cytogenetic (56.5%) and F-18 fluorodeoxyglucose positron emission tomography/computer tomography (18-FDG-PET/CT) examinations (50%) (p<0.05). ANKL is a highly aggressive leukemia with high mortality. Flow cytometry detection is sensitive for the early and differential diagnosis of ANKL with high specificity. PMID:27483437

  8. Effect of acidic pH on flow cytometric detection of bacteria stained with SYBR Green I and their distinction from background

    NASA Astrophysics Data System (ADS)

    Baldock, Daniel; Nebe-von-Caron, Gerhard; Bongaerts, Roy; Nocker, Andreas

    2013-12-01

    Unspecific background caused by biotic or abiotic particles, cellular debris, or autofluorescence is a well-known interfering parameter when applying flow cytometry to the detection of microorganisms in combination with fluorescent dyes. We present here an attempt to suppress the background signal intensity and thus to improve the detection of microorganisms using the nucleic acid stain SYBR® Green I. It has been observed that the fluorescent signals from SYBR Green I are greatly reduced at acidic pH. When lowering the pH of pre-stained samples directly prior to flow cytometric analysis, we hypothesized that the signals from particles and cells with membrane damage might therefore be reduced. Signals from intact cells, temporarily maintaining a neutral cytosolic pH, should not be affected. We show here that this principle holds true for lowering background interference, whereas the signals of membrane-compromised dead cells are only affected weakly. Signals from intact live cells at low pH were mostly comparable to signals without acidification. Although this study was solely performed with SYBR® Green I, the principle of low pH flow cytometry (low pH-FCM) might hold promise when analyzing complex matrices with an abundance of non-cellular matter, especially when expanded to non-DNA binding dyes with a stronger pH dependence of fluorescence than SYBR Green I and a higher pKa value.

  9. Glycerol-treated nuclear suspensions--an efficient preservation method for flow cytometric analysis of plant samples.

    PubMed

    Kolář, Filip; Lučanová, Magdalena; Těšitel, Jakub; Loureiro, João; Suda, Jan

    2012-02-01

    Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required. PMID:22362177

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

    PubMed

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

    2012-09-01

    Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively. PMID:22850895

  11. COUNTING CRYPTOSPORIDIUM, AN ANALYSIS OF THE UTILITY OF VARIOUS CYTOMETRIC TECHNIQUES

    EPA Science Inventory

    To develop, evaluate and implement methods to detect C. parvum oocysts in water, samples must be seeded with known concentrations of oocysts. Methods for counting oocysts are inaccurate and highly variable. To address this, several cytometric methods were tested: flow cytometry...

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

    PubMed

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

    2015-04-01

    Acute Graft-versus-Host-Disease (aGvHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (HSCT). Although rather helpful, the use of conventional immunosuppressive drugs leads to general immunosuppression and is toxic. The effects of CD4(+) T-cells, in respect to the development of aGvHD, can be altered by administration of antihuman CD4 monoclonal antibodies, here MAX.16H5 IgG1 . This approach must be tested for possible interference with the Graft-versus-Leukemia-Effect (GvL). Thus, in vitro experiments were conducted, exposing P815 leukemic cells to bone marrow and splenocytes from cd4(-/-) -C57Bl/6 mice transgenic for human CD4 and HLA-DR3 (triple transgenic mice, [TTG]) as well as previously irradiated splenocytes from Balb/c(wt) mice. Using flow cytometry, the vitality of the various malignant and graft cells was analyzed over the course of 4 days. The survival rate of P815 cells did not change significantly when exposed to MAX.16H5 IgG1 , neither did the viability of the graft cells. This provides evidence that MAX.16H5 IgG1 does not impair the GvL effect in vitro. Additionally, P815-Balb/c(wt) leukemic mice were transplanted with P815(GFP) cells, bone marrow, and splenocytes from TTG mice with and without MAX.16H5 IgG1 . Without transplantation, P815(GFP) leukemic cells could be detected by flow cytometry in the liver, the bone marrow, and the spleen of recipients. The antibodies prevented aGvHD while leaving the GvL effect intact. These findings indicate no negative effect of MAX.16H5 IgG1 on the GvL effect in vitro and in vivo after HSCT in a murine model. PMID:25717029

  13. A Single 9-Colour Flow Cytometric Method to Characterise Major Leukocyte Populations in the Rat: Validation in a Model of LPS-Induced Pulmonary Inflammation

    PubMed Central

    Barnett-Vanes, Ashton; Sharrock, Anna; Birrell, Mark A.; Rankin, Sara

    2016-01-01

    The rat is a commonly used model for immunological investigation. Yet basic research and characterisation of leukocyte populations and sub-sets lags far behind murine research, with inconsistency on reported leukocyte markers and their overlap. These shortcomings limit the opportunity for more complex and advanced rat immunology research. In this study, we developed a robust 9-colour flow-cytometric protocol to elucidate the major blood and tissue rat leukocyte populations, and validated it in a model of LPS-induced pulmonary inflammation. Blood and tissues (lung, BALF, spleen, liver, bone marrow) from naïve Sprague-Dawley rats were collected and analysed by flow cytometry (FCM). Rats were exposed to aerosolised saline or LPS (1mg/mL), at 3 and 24hrs thereafter blood, lung and BALF were collected and analysed using FCM and ELISA. Neutrophils, two monocyte subsets, NK Cells, B Cells, CD4+, CD8+ T Cells and alveolar macrophages can be identified simultaneously across different tissues using a 9-colour panel. Neutrophils and monocytes can be distinguished based upon differential expression of CD43 and His48. Neutrophils and CD43Lo/His48Hi monocyte-macrophages are elevated in the lung at 3 and 24hrs during LPS-induced pulmonary inflammation. This validated method for leukocyte enumeration will offer a platform for greater consistency in future rat immunology and inflammation research. PMID:26764486

  14. Further characterisation of the in situ terminal deoxynucleotidyl transferase (TdT) assay for the flow cytometric analysis of apoptosis in drug resistant and drug sensitive leukaemic cells

    SciTech Connect

    Chapman, R.S.; Chresta, C.M.; Herberg, A.A.

    1995-07-01

    Apoptosis, originally defined by specific morphological changes, is characterized biochemically by non-random cleavage of DNA. Depending on cell type, this DNA cleavage proceeds from 300 and 50 kbp fragments prior to, concomitantly with, or in the absence of 180 bp integer fragmentation. Incorporation into fragmented DNA of biotin-labelled nucleotides by terminal deoxynucleotidyl transferase (TdT) has recently become a standard flow cytometric assay for the identification and quantitation of apoptosis. Nucleotide incorportion is visualized using avidin-tagged fluorescein isothiocyanate (FITC). Here, we characterize this assay further in three different hemopoietic cell lines. Drug-induced DNA damage is not identified by the TdT assay unless it is coupled to the apoptotic response. This was demonstrated using cells in which activation of the oncogenic Abelson-encoded protein tyrosine kinase suppressed drug-induced apoptosis, but did not inhibit drug-induced DNA damage (by melphalan, hydroxyurea, or etoposide). Furthermore, the TdT assay identifies DNA fragments formed during apoptosis induced by etoposide and N-methylformamide in HL60 and MOLT-4 cells, including those high molecular weight DNA fragments formed in MOLT-4 cells which were not further cleaved to 180-200 bp integer fragments. Our results support the use of flow cytometry and the TdT assay to reliably measure apoptotic cells in heterogeneous cell samples. 55 refs., 7 figs., 2 tabs.

  15. Flow Cytometric Analysis of BrdU Incorporation as a High-Throughput Method for Measuring Adult Neurogenesis in the Mouse

    PubMed Central

    Balu, Darrick T.; Hodes, Georgia E.; Hill, Tiffany E.; Ho, Nancy; Rahman, Zia; Bender, Corey N.; Ring, Robert H.; Dwyer, Jason M.; Rosenzweig-Lipson, Sharon; Hughes, Zoe A.; Schechter, Lee E.; Lucki, Irwin

    2009-01-01

    Introduction The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze. Methods Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day. Results The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells. Discussion These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology. PMID:19121403

  16. Flow Cytometric Detection of Mycobacterium avium subsp. paratuberculosis-Specific Antibodies in Experimentally Infected and Naturally Exposed Calves

    PubMed Central

    Bridger, P. S.; Bulun, H.; Fischer, M.; Akineden, Ö.; Seeger, T.; Barth, S.; Henrich, M.; Doll, K.; Bülte, M.; Menge, C.; Bauerfeind, R.

    2013-01-01

    A desirable test to diagnose infections with Mycobacterium avium subsp. paratuberculosis facilitates identification of infected cattle prior to the state of M. avium subsp. paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. avium subsp. paratuberculosis bacteria as the antigen, for diagnosis of M. avium subsp. paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. avium subsp. paratuberculosis shedders, 22 M. avium subsp. paratuberculosis free). After preadsorption with Mycobacterium phlei, sera were incubated with M. avium subsp. paratuberculosis and M. avium subsp. avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. avium subsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. avium subsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. avium subsp. paratuberculosis infections rather than methodological constraints. PMID:23885032

  17. Establishment of a microplate assay for flow cytometric assessment and it is use for the evaluation of age-related phenotypic changes in canine whole blood leukocytes.

    PubMed

    Reis, Alexandre B; Carneiro, Cláudia M; Carvalho, Maria das Graças; Teixeira-Carvalho, Andréa; Giunchetti, Rodolfo C; Mayrink, Wilson; Genaro, Odair; Corrêa-Oliveira, Rodrigo; Martins-Filho, Olindo A

    2005-02-10

    The effectiveness of flow cytometric assays for canine use is still requiring standardization. Despite several studies using purified mononuclear cells, no methodology or reference ranges are available for immunophenotyping of whole blood leukocytes (WBL). Fresh and pre-fixed WBL were used to identify cell-subsets, (Thy-1(+)/CD5(+)/CD4(+)/CD8(+)/CD21(+) and CD14(+)) and measure MHC-II, CD45RA/CD45RB expression. We described here an efficient method for fast quantification of canine-WBL, using pre-fix in a microplate assay, which allows long-term sample storage prior to phenotyping. Decreased percentage of CD5(+)-T-cells within the lymphocyte-gate and increased percentage of CD21(+)-B-cells were observed in young animals, which led to higher T/B cell ratios in middle-aged dogs. Lower numerical counts of Thy-1(+), CD4(+), CD8(+) and CD21(+) lymphocyte were observed when compared to young animals. In addition, we identified an age-related decline of MHC-II/CD45RA expression by lymphocytes. We proposed an improved method for phenotyping of canine peripheral blood mononuclear cells (PBMC) that has significant use for researchers and veterinary clinicians. The hematological changes of senescence previously identified on PBMC could be adequately reproduced on features identified by whole blood. Furthermore, this study supplies normal range references as baseline standards for clinical purposes, besides specific immunological parameters to monitor canine aging process. PMID:15621304

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

    PubMed Central

    Labedz-Maslowska, Anna; Kamycka, Elzbieta; Bobis-Wozowicz, Sylwia; Madeja, Zbigniew; Zuba-Surma, Ewa K.

    2016-01-01

    Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury. PMID:26633976

  19. Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

    PubMed Central

    Ochyl, Lukasz J.; Moon, James J

    2015-01-01

    Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a “pathogen-mimicking” nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles. PMID:25992469

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

    PubMed Central

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

    2015-01-01

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

  1. Residual normal stem cells can be detected in newly diagnosed chronic myeloid leukemia patients by a new flow cytometric approach and predict for optimal response to imatinib.

    PubMed

    Janssen, J J W M; Deenik, W; Smolders, K G M; van Kuijk, B J; Pouwels, W; Kelder, A; Cornelissen, J J; Schuurhuis, G J; Ossenkoppele, G J

    2012-05-01

    Insensitivity of chronic myeloid leukemia (CML) hematopoietic stem cells to tyrosine kinase inhibitors (TKIs) prevents eradication of the disease and may be involved in clinical resistance. For improved treatment results more knowledge about CML stem cells is needed. We here present a new flow cytometric approach enabling prospective discrimination of CML stem cells from their normal counterparts within single-patient samples. In 24 of 40 newly diagnosed CML patients residual normal CD34(+)CD38(-) stem cells could be identified by lower CD34 and CD45 expression, lower forward/sideward light scatter and by differences of lineage marker expression (CD7, CD11b and CD56) and of CD90. fluorescent in situ hybridization (FISH) analysis on Fluorescence-activated cell sorting sorted cells proved that populations were BCR-ABL positive or negative and long-term liquid culture assays with subsequent colony forming unit assays and FISH analysis proved their stem cell character. Patients with residual non-leukemic stem cells had lower clinical risk scores (Sokal, Euro), lower hematological toxicity of imatinib (IM) and better molecular responses to IM than patients without. This new approach will expand our possibilities to separate CML and normal stem cells, present in a single bone marrow or peripheral blood sample, thereby offering opportunities to better identify new CML stem-cell-specific targets. Moreover, it may guide optimal clinical CML management. PMID:22157734

  2. Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia

    SciTech Connect

    Arkesteijn, G.J.A.; Erpelinck, S.L.A.; Martens, A.C.M.; Hagenbeek, A.

    1995-04-01

    Flow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde-fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin- and DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120-fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000. 23 refs., 3 figs., 3 tabs.

  3. A clinically applicable method to preserve urine and bladder washing cells for flow cytometric monitoring of bladder cancer patients.

    PubMed

    Deitch, A D; Andreotti, V A; Strand, M A; Howell, L; deVere White, R W

    1990-04-01

    We describe a method to fix exfoliated bladder cells that is suitable for followup of bladder cancer patients by deoxyribonucleic acid flow cytometry. After fixation with room temperature methanol plus acetic acid (20:1, volume:volume) urine and bladder washing samples from these patients can be stored at room temperature for 3 to 7 days and then assessed reliably for the presence of aneuploidy and the percentage of hyperdiploid cells. For those with active transitional cell carcinoma diagnostic accuracy comparing fresh to fixed specimens was improved from 58 to 92% with urine and from 50 to 100% with washing samples. For patients with a history of transitional cell carcinoma who currently are free of disease the false positive rate remains unchanged after fixation. The procedure described is suitable for use in the outpatient clinic and should permit shipping of samples without refrigeration to a central flow cytometry facility for analysis. PMID:2179581

  4. Flow cytometric and near-infrared Raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm.

    PubMed

    Li, Xiao-Xia; Wang, Meng; Chen, Huan-Hua; Li, Qing-Yang; Yang, Huan; Xu, Hui-Yan; Lu, Yang-Qing; Zhang, Ming; Yang, Xiao-Gan; Lu, Sheng-Sheng; Lu, Ke-Huan

    2016-07-01

    Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen-thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality. PMID:27095613

  5. Flow cytometric analysis of mast cells from normal and pathological human bone marrow samples: identification and enumeration.

    PubMed Central

    Orfao, A.; Escribano, L.; Villarrubia, J.; Velasco, J. L.; Cerveró, C.; Ciudad, J.; Navarro, J. L.; San Miguel, J. F.

    1996-01-01

    In the present paper we have used a three-color immunofluorescence procedure combined with flow cytometry cell analysis and sorting for the identification and enumeration of human mast cells in both normal and pathological bone marrow samples. Our results show that bone marrow mast cells are clearly identifiable on the basis of their light-scatter properties and strong CD117 expression. These cells were negative for the CD34, CD38, and BB4 antigens. In addition, they were CD33+ and displayed a high reactivity for the anti-IgE monoclonal antibody. The identity of the CD117-strong+ cells (mast cells) was confirmed by both microscopic examination and flow cytometry analysis. The overall frequency of mast cells in the bone marrow samples analyzed in the present study was constantly lower than 1%. The lowest frequencies corresponded to normal human bone marrow samples (0.0080 +/- 0.0082%) and the highest to those patients suffering from indolent systemic mast cell disease (0.40 +/- 0.13%). In summary, our results show that the identification and enumeration of bone marrow mast cells can be achieved using multiparametric flow cytometry. Moreover, once identified, mast cells are suitable for being characterized from the phenotypic and the functional point of view, facilitating the comparison between normal and abnormal mast cells. Images Figure 3 PMID:8909239

  6. Oviduct fluid and heparin induce similar surface changes in bovine sperm during capacitation: a flow cytometric study using lectins.

    PubMed

    Mahmoud, A I; Parrish, J J

    1996-04-01

    Eight different lectins conjugated to fluorescein isothiocyanate (FITC) were used to screen for sperm plasma membrane changes during in vitro capacitation of bovine sperm. Analysis of lectin binding to sperm was done using flow cytometry. Of the eight lectins, only Triticum vulgaris (wheat germ agglutinin, WGA) binding to sperm was altered with capacitation. Capacitation of bovine sperm by heparin was found to decrease WGA binding to sperm by 78% (P < 0.05). The effect of capacitation by oviduct fluid was next compared with capacitation by heparin for changes in WGA binding to sperm. The effect of inhibiting capacitation with glucose on WGA binding was also determined. WGA-bound sperm were detected by flow cytometry as being present in two fluorescence peaks defined as low fluorescence (A) or high fluorescence (B) intensity. The percentage of sperm in peak A was greater for heparin and oviduct fluid-treated sperm compared to sperm incubated under noncapacitating conditions in only culture medium (P < 0.001). Capacitation with either heparin or oviduct fluid was inhibited by glucose as assessed by the ability of lysophosphatidylcholine (100 micrograms/ml) to induce acrosome reactions. Glucose also reduced the percentage of sperm in peak A for both heparin- and oviduct fluid-treated sperm (P < 0.01). We conclude that heparin or oviduct fluid induced changes on the sperm plasma membrane during capacitation. Binding sites for WGA on sperm were either structurally altered or lost during capacitation. PMID:9052948

  7. Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia.

    PubMed

    Bihari, Nevenka; Micić, Milena; Batel, Renato; Zahn, Rudolf K

    2003-07-16

    Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants. PMID:12799105

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

    PubMed

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

    2013-03-01

    In this study, we investigate the validity and laboratory utility of flow cytometry when analyzing platelet activation by studying CD41, CD42b, CD62P and CD63. We compare flow cytometry results from citrated whole-blood and finger-prick samples directly after collection and also after storing both a finger-prick and whole-blood sample for 24 hours. Citrated whole-blood and finger-prick samples were taken from three healthy individuals on two occasions, and a total of 60,000 cells were analyzed for each of the four phycoerythrin-labeled monoclonal antibodies. Half of each sample was analyzed immediately after sampling while the other half was kept in the fridge at 6 °C for 24 hours before analysis. No significant difference was found between the sampling methods or the period of time before analysis. Results therefore suggest that an appropriately prepared finger-prick sample can be used for platelet function analysis, and samples can be stored for 24 hours in the fridge at 6 °C before analysis. PMID:23320994

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

    PubMed

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

    2014-02-01

    The combined effects of DO and TiO2 (mixed rutile/anatase phase, 7/3) on immune responses in Perna viridis were examined. Mussels were exposed to six combinations of oxygen levels (hypoxia: 1.5 mg O2l(-1), normoxia: 6.0 mg O2 l(-1)) and TiO2 concentrations (0, 2.5 mg l(-1) and 10 mg l(-1)) for 216 h. Mussels were sampled after 24h, 48h, 120 h and 216 h, and immune parameters of hemocytes, including mortality, phagocytosis, non-specific esterase, ROS production, lysosomal content and total hemocyte count were investigated using flow cytometric assay. Hemocyte mortality was higher under hypoxia than normoxia, and increased with TiO2 concentrations, but no interaction was found between DO and TiO2. Phagocytosis was reduced under hypoxia and decreased with TiO2 exposure, and the interactive effect between time and TiO2 was observed. The percentage of hemocytes showing non-specific esterase activity was lower under hypoxia, and decreased as TiO2 concentration increased with the significant interactive effect of DO and TiO2. ROS production and lysosomal content were lower under hypoxia and reduced as concentration of TiO2 increased, and interactive effect of DO and TiO2 on ROS was evident. THC was significantly affected by the interactive effect between TiO2 and DO, with higher values under normoxia in the presence of TiO2. The present study demonstrated that immune functions of P. viridis were influenced by both nano-TiO2 and hypoxia with some synergistic effects between the two stressors. This implies that DO has to be considered in the evaluation of the toxicity of nano-materials to bivalves. PMID:24189102

  10. High-speed flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood: preliminary in-vitro studies

    NASA Astrophysics Data System (ADS)

    Cooper, Christy L.; Leary, James F.

    2014-03-01

    Leukemic cancer stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in peripheral blood of leukemia patients. The leukemic stem cells are also highly resistant to standard chemotherapeutic regimens so new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial studies we have designed an antibody-targeted and fluorescent (Cy5.5) nanoparticle for targeting these leukemic stem cells and then introducing new strategies for killing them. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell line RS4;11 (with putative immunophenotype CD123+/CD24+/CD38-/CD10-/Flt-3-) was used as a model human leukemic stem cell systems and were spiked into normal human peripheral blood cells containing normal blood stem-progenitor cells (immunophenotype CD123-/CD34+/CD38-) and Cy5.5-labeled nanoparticles with targeting molecule anti-CD123 antibody. An irrelevant antibody (CD71) which should not bind to any live leukemic stem cell or normal stem cell (binds erythrocytes) was used as a way of distinguishing between true-positive live and false-positive damaged/dead cells, the latter occurring at much higher frequencies than the very rare (e.g. 0.001 to 0.0001 percent frequency true leukemic stem cells). These studies are designed to measure the targeting sensitivity and specificity of the fluorescent nanoparticles to the putative rare leukemic stem cells with the eventual design to use the nanoparticles to direct killing therapeutic doses to the leukemic stem cells but not to the normal stem-progenitor cells.

  11. Advanced flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood in a defined model system

    NASA Astrophysics Data System (ADS)

    Cooper, Christy L.; Leary, James F.

    2015-03-01

    Leukemia stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in the peripheral blood of leukemia patients. Since leukemic stem cells are also resistant to standard chemotherapeutic regimens, new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial targeting studies we utilized a bioinformatics approach to design an antibodyfluorescent nanoparticle conjugate for targeting to these leukemic stem cells and to minimize targeting to normal stemprogenitor cells. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell RS4;11 (with putative immunophenotype CD133+/CD24+/-, CD34+/-, CD38+, CD10-/Flt3+) was spiked into normal hematopoietic stem-progenitor cells obtained from a "buffy coat" prep (with putative immunophenotype CD133- /CD34+/CD38-/CD10-/Flt-3-) to be used as a model human leukemia patient. To analyze the model system, digital data mixtures of the two cell types were first created and assigned classifiers in order to create truth sets. ROC (Receiver Operating Characteristic) and multidimensional cluster analyses were used to evaluate the specificity and sensitivity of the immunophenotyping panel and for automated cell population identification, respectively. Costs of misclassification (false targeting) were also accounted for by this analysis scheme. Ultimately, this analysis scheme will be applied to use of nanoparticle-antibody conjugates at therapeutic doses for targeted killing of leukemia stem cells preferentially to normal stem -progenitor cells.

  12. Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

    PubMed

    Trávníček, Pavel; Ponert, Jan; Urfus, Tomáš; Jersáková, Jana; Vrána, Jan; Hřibová, Eva; Doležel, Jaroslav; Suda, Jan

    2015-10-01

    Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species. PMID:25929591

  13. Effects of high ambient temperature on fish sperm plasma membrane integrity and mitochondrial activity - A flow cytometric study.

    PubMed

    Nagy, Szabolcs Tamás; Kakasi, Balázs; Pál, László; Havasi, Máté; Bercsényi, Miklós; Husvéth, Ferenc

    2016-06-01

    Local extreme climatic conditions occurring as a result of global climate change may interfere with the reproduction of animals. In the present study fish spermatozoa were incubated at different temperatures (20, 25, 30 and 40 °C) for 10 and 30 minutes, respectively and plasma membrane integrity and mitochondrial membrane potential changes were evaluated with flow cytometry using SYBR-14/PI and Mitotracker Deep Red FM fluorescent dyes. No significant differences were found in plasma membrane integrity at either incubation temperatures or time points. Mitotracker Deep Red FM histogram profiles indicating mitochondrial activity showed significant (p < 0.001) alterations in all cases of higher (25, 30 and 40 °C) temperature treatments as compared to the samples incubated at 20 °C. Our results indicate that fish spermatozoa exposed to high temperatures suffer sublethal damage that cannot be detected with conventional, vital staining techniques. PMID:27165524

  14. Engineering and Flow-Cytometric Analysis of Chimeric LAGLIDADG Homing Endonucleases from Homologous I-OnuI-Family Enzymes

    PubMed Central

    Baxter, Sarah K.; Scharenberg, Andrew M.; Lambert, Abigail R.

    2014-01-01

    LAGLIDADG homing endonucleases (LHEs) are valuable tools for genome engineering, and our ability to alter LHE target site specificity is rapidly evolving. However, widespread use of these enzymes is limited due to the small number of available engineering scaffolds, each requiring extensive redesign to target widely varying DNA sequences. Here, we describe a technique for the chimerization of homologous I-OnuI family LHEs. Chimerization greatly expands the pool of unique starting scaffolds, thereby enabling more effective and efficient LHE redesign. I-OnuI family enzymes are divided into N- and C-terminal halves based on sequence alignments, and then combinatorially rejoined with a hybrid linker. The resulting chimeric enzymes are expressed on the surface of yeast where stability, DNA binding affinity, and cleavage activity can be assayed by flow cytometry. PMID:24510269

  15. Comparison of in vitro and in vivo clastogenic potency based on benchmark dose analysis of flow cytometric micronucleus data.

    PubMed

    Bemis, Jeffrey C; Wills, John W; Bryce, Steven M; Torous, Dorothea K; Dertinger, Stephen D; Slob, Wout

    2016-05-01

    The application of flow cytometry as a scoring platform for both in vivo and in vitro micronucleus (MN) studies has enabled the efficient generation of high quality datasets suitable for comprehensive assessment of dose-response. Using this information, it is possible to obtain precise estimates of the clastogenic potency of chemicals. We illustrate this by estimating the in vivo and the in vitro potencies of seven model clastogenic agents (melphalan, chlorambucil, thiotepa, 1,3-propane sultone, hydroxyurea, azathioprine and methyl methanesulfonate) by deriving BMDs using freely available BMD software (PROAST). After exposing male rats for 3 days with up to nine dose levels of each individual chemical, peripheral blood samples were collected on Day 4. These chemicals were also evaluated for in vitro MN induction by treating TK6 cells with up to 20 concentrations in quadruplicate. In vitro MN frequencies were determined via flow cytometry using a 96-well plate autosampler. The estimated in vitro and in vivo BMDs were found to correlate to each other. The correlation showed considerable scatter, as may be expected given the complexity of the whole animal model versus the simplicity of the cell culture system. Even so, the existence of the correlation suggests that information on the clastogenic potency of a compound can be derived from either whole animal studies or cell culture-based models of chromosomal damage. We also show that the choice of the benchmark response, i.e. the effect size associated with the BMD, is not essential in establishing the correlation between both systems. Our results support the concept that datasets derived from comprehensive genotoxicity studies can provide quantitative dose-response metrics. Such investigational studies, when supported by additional data, might then contribute directly to product safety investigations, regulatory decision-making and human risk assessment. PMID:26049158

  16. Flow Cytometric Testing of Green Fluorescent Protein-Tagged Lactobacillus rhamnosus GG for Response to Defensins

    PubMed Central

    De Keersmaecker, Sigrid C. J.; Braeken, Kristien; Verhoeven, Tine L. A.; Perea Vélez, Mónica; Lebeer, Sarah; Vanderleyden, Jos; Hols, Pascal

    2006-01-01

    Lactobacillus rhamnosus GG is of general interest as a probiotic. Although L. rhamnosus GG is often used in clinical trials, there are few genetic tools to further determine its mode of action or to develop it as a vehicle for heterologous gene expression in therapy. Therefore, we developed a reproducible, efficient electroporation procedure for L. rhamnosus GG. The best transformation efficiency obtained was 104 transformants per μg of DNA. We validated this protocol by tagging L. rhamnosus GG with green fluorescent protein (GFP) using the nisin-controlled expression (NICE) system. Parameters for overexpression were optimized, which allowed expression of gfp in L. rhamnosus GG upon induction with nisin. The GFP+ strain can be used to monitor the survival and behavior of L. rhamnosus GG in vivo. Moreover, implementation of the NICE system as a gene expression switch in L. rhamnosus GG opens up possibilities for improving and expanding the performance of this strain. The GFP-labeled strain was used to demonstrate that L. rhamnosus GG is sensitive to human beta-defensin-2 but not to human beta-defensin-1. PMID:16820489

  17. Evaluating the Safety of Somatic Periosteal Cells by Flow-Cytometric Analysis Monitoring the History of DNA Damage.

    PubMed

    Kawase, Tomoyuki; Hayama, Kazuhide; Tsuchimochi, Makoto; Nagata, Masaki; Okuda, Kazuhiro; Yoshie, Hiromasa; Burns, Douglas M; Nakata, Koh

    2016-04-01

    In preparing cell-based products for regenerative therapy, cell quality should be strictly controlled. Methodologies for evaluating cell viability, identity, and purity are established and used routinely, whereas current methodologies for evaluating cell safety, particularly genetic integrity or tumorigenicity, are time-consuming and relatively insensitive. As part of developing a more practical screening system, the authors previously demonstrated that γ-H2AX and p53 were useful markers for evaluating the history of DNA damage. To validate these markers further and develop a more quantitative methodology, single cell-based expression of these markers and two additional candidates have now been examined using flow cytometry (FCM). FCM analysis and immunofluorescent staining demonstrated that γ-ray-irradiation suppressed proliferation, enlarged cells, and cell nuclei, and immediately upregulated γ-H2AX and p21(waf1) in large numbers of cells for up to 12 days. Gamma-H2AX foci were formed in the nuclei of many affected cells. An initial sharp increase in p53 expression declined slowly over 12 days, while Rb expression increased linearly. The present findings suggest that this high-throughput, cell-based, combinational evaluation of protein markers and cell size enables a small number of cells with a history of DNA damage to be detected quickly and routinely from within a very large cell population. Using this screening methodology will improve the ability to verify the quality of cell-based products used in regenerative therapy. PMID:26828697

  18. Flow-cytometric cell sorting and subsequent molecular analyses for culture-independent identification of bacterioplankton involved in dimethylsulfoniopropionate transformations.

    PubMed

    Mou, Xiaozhen; Moran, Mary Ann; Stepanauskas, Ramunas; González, José M; Hodson, Robert E

    2005-03-01

    Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 microM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, alpha-Proteobacteria); Caulobacter (alpha-Proteobacteria); and Brachymonas and Xenophilus (beta-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, alpha-Proteobacteria) and novel gamma-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally. PMID:15746343

  19. Optimized flow cytometric assay for the measurement of platelet microparticles in plasma: pre-analytic and analytic considerations.

    PubMed

    Kim, H K; Song, K S; Lee, E S; Lee, Y J; Park, Y S; Lee, K R; Lee, S N

    2002-07-01

    Platelet microparticles (PMP) are submicroscopic membrane vesicles released by platelets during activation. Flow cytometry is the most widely used method for quantifying PMP, but the optimization of the technical method has not yet been fully evaluated. This study was designed to assess the pre-analytical variables including blood sampling conditions, and to evaluate the analytical variations including effect of the platelet-specific antibodies and quantitative beads, precision, linearity and accuracy in comparison with beta-thromboglobulin, which is one of the platelet activation markers. Numbers of PMP collected into citrate-theophylline-adenosine-dipyridamole (CTAD) tubes were increased with time, but to a lesser extent than when collected into sodium citrate tubes. The precision of the PMP assay was relatively high. Excellent linear correlation was observed for dilution linearity. Regarding the platelet-specific antibodies used, anti-CD41a-labeled samples resulted in higher PMP levels than those labeled with anti-CD61 and anti-CD42a. There was no significant difference of PMP counts according to the quantitative beads. The PMP assay is well correlated with beta-thromboglobulin levels. Our findings suggest that blood samples for the PMP assay should be collected in a CTAD tube and delayed measurement is not allowed to avoid artefactual platelet activation. The PMP assay can be used successfully as a useful marker of the detection of in vivo platelet activation, provided that pre-analytical and technical points are optimally taken into consideration. PMID:12138366

  20. Flow-Cytometric Cell Sorting and Subsequent Molecular Analyses for Culture-Independent Identification of Bacterioplankton Involved in Dimethylsulfoniopropionate Transformations

    PubMed Central

    Mou, Xiaozhen; Moran, Mary Ann; Stepanauskas, Ramunas; González, José M.; Hodson, Robert E.

    2005-01-01

    Marine bacterioplankton transform dimethylsulfoniopropionate (DMSP) into the biogeochemically important and climatically active gas dimethylsulfide. In order to identify specific bacterial taxa mediating DMSP processing in a natural marine ecosystem, we amended water samples from a southeastern U.S. salt marsh with 20 μM DMSP and tracked community shifts with flow cytometry (FCM) coupled to 16S rRNA gene analyses. In two out of four seasons studied, DMSP amendments induced the formation of distinct bacterioplankton populations with elevated nucleic acid (NA) content within 24 h, indicative of cells actively utilizing DMSP. The 16S rRNA genes of the cells with and without elevated NA content were analyzed following cell sorting and PCR amplification with sequencing and terminal restriction fragment length polymorphism approaches. Compared to cells in the control FCM populations, bacteria with elevated NA content in the presence of DMSP were relatively enriched in taxa related to Loktanella, Oceanicola, and Sulfitobacter (Roseobacter lineage, α-Proteobacteria); Caulobacter (α-Proteobacteria); and Brachymonas and Xenophilus (β-Proteobacteria) in the May-02 sample and to Ketogulonicigenium (Roseobacter lineage, α-Proteobacteria) and novel γ-Proteobacteria in the Sept-02 sample. Our study suggests that diverse bacterioplankton participate in the metabolism of DMSP in coastal marine systems and that their relative importance varies temporally. PMID:15746343

  1. Flow cytometric analysis of FSHR, BMRR1B, LHR and apoptosis in granulosa cells and ovulation rate in merino sheep.

    PubMed

    Regan, Sheena L P; McFarlane, James R; O'Shea, Tim; Andronicos, Nicholas; Arfuso, Frank; Dharmarajan, Arun; Almahbobi, Ghanim

    2015-08-01

    The aim of the present study was to determine the direct cause of the mutation-induced, increased ovulation rate in Booroola Merino (BB) sheep. Granulosa cells were removed from antral follicles before ovulation and post-ovulation from BB (n=5) and WT (n=12) Merino ewes. Direct immunofluorescence measurement of mature cell surface receptors using flow cytometry demonstrated a significant up-regulation of FSH receptor (FSHR), transforming growth factor beta type 1, bone morphogenetic protein receptor (BMPR1B), and LH receptor (LHR) in BB sheep. The increased density of FSHR and LHR provide novel evidence of a mechanism for increasing the number of follicles that are recruited during dominant follicle selection. The compounding increase in receptors with increasing follicle size maintained the multiple follicles and reduced the apoptosis, which contributed to a high ovulation rate in BB sheep. In addition, we report a mutation-independent mechanism of down-regulation to reduce receptor density of the leading dominant follicle in sheep. The suppression of receptor density coincides with the cessation of mitogenic growth and steroidogenic differentiation as part of the luteinization of the follicle. The BB mutation-induced attenuation of BMPR1B signaling led to an increased density of the FSHR and LHR and a concurrent reduction in apoptosis to increase the ovulation rate. The role of BMPs in receptor modulation is implicated in the development of multiple ovulations. PMID:25948249

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

    PubMed

    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). PMID:25483368

  3. Flow cytometric analysis of circulating endothelial cells and endothelial progenitors for clinical purposes in oncology: A critical evaluation

    PubMed Central

    DANOVA, MARCO; COMOLLI, GIUDITTA; MANZONI, MARIANGELA; TORCHIO, MARTINA; MAZZINI, GIULIANO

    2016-01-01

    Malignant tumors are characterized by uncontrolled cell growth and metastatic spread, with a pivotal importance of the phenomenon of angiogenesis. For this reason, research has focused on the development of agents targeting the vascular component of the tumor microenvironment and regulating the angiogenic switch. As a result, the therapeutic inhibition of angiogenesis has become an important component of anticancer treatment, however, its utility is partly limited by the lack of an established methodology to assess its efficacy in vivo. Circulating endothelial cells (CECs), which are rare in healthy subjects and significantly increased in different tumor types, represent a promising tool for monitoring the tumor clinical outcome and the treatment response. A cell population circulating into the blood also able to form endothelial colonies in vitro and to promote vasculogenesis is represented by endothelial progenitor cells (EPCs). The number of both of these cell types is extremely low and they cannot be identified using a single marker, therefore, in absence of a definite consensus on their phenotype, require discrimination using combinations of antigens. Multiparameter flow cytometry (FCM) is ideal for rapid processing of high numbers of cells per second and is commonly utilized to quantify CECs and EPCs, however, remains technically challenging since there is as yet no standardized protocol for the identification and enumeration of these rare events. Methodology in studies on CECs and/or EPCs as clinical biomarkers in oncology is heterogeneous and data have been obtained from different studies leading to conflicting conclusions. The present review presented a critical review of the issues that limit the comparability of results of the most significant studies employing FCM for CEC and/or EPC detection in patients with cancer. PMID:27284422

  4. LIF is Essential for SVZ Neural Stem Cell and Progenitor Homeostasis as Revealed by a Novel Flow Cytometric Analysis

    PubMed Central

    Buono, Krista D.; Vadlamuri, Daimler; Gan, Qiong; Levison, Steven W.

    2013-01-01

    Stem cells rely on extracellular signals produced by the niche, which dictate their ability to self-renew, expand and differentiate. It is essential to have sensitive and reproducible methods of either quantifying or isolating these stem cells and progenitors to understand their intrinsic properties and how extrinsic signals regulate their development. However, stem cells are difficult to distinguish from multipotential progenitors, which may look and act like them. Here we define a 4-color flow cytometry panel using CD133, LeX, CD140a, NG2 to define an NSC as well as 4 classes of multipotential progenitors and 3 classes of bipotential progenitors, several of which have not been previously described. We performed gain and loss of function studies for LIF and show a depletion of NSCs, a subset of multipotential neural precursors and immature oligodendrocytes in LIF null mice. Gain of function studies showed that LIF increased the abundance of these precursors. Our studies also show that these NPs have differential requirements for LIF and CNTF and for EGF, FGF-2 and PDGF for their propagation in vitro. Surprisingly, the related cytokine, CNTF was less potent than LIF in increasing the NSCs and more potent than LIF in increasing the PDGF responsive multipotential precursors. Finally, we show that LIF increases the expression of the core transcription factors: Klf4, Fbx15, Nanog, Sox2 and c-Myc. Altogether our FACS analyses reveal that the neonatal SVZ is far more heterogeneous than previously suspected and our studies provide new insights into the signals and mechanisms that regulate their self-renewal and proliferation. PMID:23258129

  5. Flow cytometric characterizations of leukocyte subpopulations in the peripheral blood of northern pig-tailed macaques (Macaca leonina)

    PubMed Central

    ZHENG, Hong-Yi; ZHANG, Ming-Xu; ZHANG, Lin-Tao; ZHANG, Xiao-Liang; PANG, Wei; LYU, Long-Bao; ZHENG, Yong-Tang

    2014-01-01

    Pig-tailed macaques (Macaca nemistrina group) have been extensively used as non-human primate animal models for various human diseases in recent years, notably for AIDS research due to their sensitivity to HIV-1. Northern pig-tailed macaques (M. leonina) are distributed in China and other surrounding Southeast Asia countries. Although northern pig-tailed macaques have been bred on a large scale as experimental animals since 2012, the reference value of normal levels of leukocytes is not available. To obtain such information, 62 blood samples from male and female healthy northern pig-tailed macaques at different ages were collected. The normal range of major leukocyte subpopulations, such as T lymphocytes, B lymphocytes, natural killer (NK) cells, monocytes, and the expression levels of activation or differentiation related molecules (CD38, HLA-DR, CCR5, CD21, IgD, CD80 and CD86) on lymphocytes were analyzed by flow cytometry. The counts of B cells decreased with age, but those of CD8+ T cells and NK cells and the frequency of CD38+HLA-DR+CD4+ T cells were positively correlated with age. The counts of leukocyte subpopulations were higher in males than those in females except for CD4+ T cells. Males also showed higher expression levels of IgD and CD21 within B cells. This study provides basic data about the leukocyte subpopulations of northern pig-tailed macaques and compares this species with commonly used Chinese rhesus macaques (M. mulatta), which is meaningful for the biomedical application of northern pig-tailed macaques. PMID:25465082

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

    NASA Astrophysics Data System (ADS)

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

    2012-05-01

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

  7. Flow cytometric immunophenotyping of feline bone marrow cells and haematopoietic progenitor cells using anti-human antibodies.

    PubMed

    Araghi, Atefeh; Nassiri, Seyed Mahdi; Atyabi, Nahid; Rahbarghazi, Reza; Mohammadi, Elham

    2014-04-01

    There is a paucity of species-specific antibodies available for feline haematopoietic conditions. The purpose of this study was to broaden the panel of antibodies available for use in the immunophenotypic characterisation of feline haematopoietic cells by testing clones of anti-human monoclonal antibodies (mAbs) on normal, neoplastic and cultured feline haematopoietic progenitors to determine cross-reactivity to feline counterparts. In this study, 24 clones of anti-human mAbs were tested on normal or neoplastic feline bone marrow and peripheral blood cells. Six of these mAbs, including anti-cluster of differentiation (CD)61, anti-CD18, anti-CD14, anti-CD235a, anti-CD41 and anti-CD29, cross-reacted with normal feline bone marrow cells, whereas anti-CD33 and anti-CD117 cross-reacted with the blast cells in the bone marrow of two cats with myelodysplastic syndrome, and anti-CD71, anti-235a, anti-41 and anti-42 cross-reacted with immature erythroid cells in a cat with erythroleukaemia. In a feline immunodeficiency virus-positive cat, bone marrow cells were labelled with anti-CD33, anti-14 and anti-45. Anti-CD18, anti-CD14, anti-CD41 and anti-CD61 also reacted with the peripheral blood cells of the healthy cats. The feline haematopoietic progenitors formed colonies in the methylcellulose-based semisolid medium with significant enrichment of colony-forming unit-granulocyte, monocyte and burst-forming unit-erythroid. A panel of six anti-feline mAbs (anti-CD21-like, anti-T lymphocytes, anti-CD172a, anti-granulocyte, anti-CD45-like and anti-CD18) and eight anti-human antibodies (anti-CD71, anti-CD33, anti-CD235a, anti-CD41, anti-CD61, anti-CD117, anti-CD38 and anti-CD34) were used for the immunophenotypic characterisation of the feline bone marrow progenitors. CD45, CD33, CD235a and CD18 were expressed by the feline haematopoietic progenitor cells, with the highest expression level for CD45. PMID:24065708

  8. Handling of boar spermatozoa during and after flow cytometric sex-sorting process to improve their in vitro fertilizing ability.

    PubMed

    del Olmo, D; Parrilla, I; Gil, M A; Maside, C; Tarantini, T; Angel, M A; Roca, J; Martinez, E A; Vazquez, J M

    2013-09-01

    The objective of this study was to develop an adequate sperm handling protocol in order to obtain a sex-sorted sperm population with an optimal fertilizing ability. For this purpose, different aspects of the sorting procedure were examined. The effects of the high dilution rates (experiment 1), type of collection medium used (experiment 2), and sheath fluid composition (experiment 3) on sorted boar sperm quality and function were evaluated. Sperm quality was assessed by motility and viability tests, whereas sperm function was evaluated by an in vitro fertilization assay which determined the penetration and polyspermy rates as well as the mean number of sperm penetrating each oocyte. In experiment 1, the results obtained indicated that the high dilution rates did not cause a decrease either in the sperm quality parameters evaluated or the in vitro fertilization ability of spermatozoa. In experiment 2, although sperm quality was not affected, fertilizing ability was compromised after sorting, regardless of the collection medium that was used. In the experiment 3, all groups displayed adequate sperm quality values, but higher in vitro fertility parameters were obtained for spermatozoa sorted in presence of EDTA in the sheath fluid and egg yolk (EY) in the collection media when compared with those sorted in absence of these protective agents. No differences in penetration rates between unsorted highly diluted (control) and sorted sperm in the presence of EDTA and EY were observed. In conclusion, fertilizing ability was compromised in sex-sorted sperm. The addition of EDTA to sheath fluid and EY to collection medium improved boar sperm fertilizing ability, and both agents should be included as essential media components in future studies. PMID:23746874

  9. Cytometric patterns reveal growth states of Shewanella putrefaciens

    PubMed Central

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

    2015-01-01

    Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species. PMID:25185955

  10. Fluorescent Particles For Flow Testing

    NASA Technical Reports Server (NTRS)

    Bonnell, Jeremy L.; Stern, Susan M.; Torkelson, Jan R.

    1995-01-01

    Small alumina spheres coated with fluorescent dye used in flow testing of transparent plastic model of check valve. Entrained fluroescent particles make flows visible. After completion of flow test, particles remaining in valve easily detectable and removed for measurement of their sizes.