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PubMedDate not supplied

A simple method for counting small numbers of cells by flow cytometry.

We describe a flow cytometric method for the quantitation of small numbers of cells that facilitates proliferation studies in microcultures. A constant number of fluorescent latex microspheres/sample is added to single cell suspensions prepared from the cultures. By flow cytometric analysis, cells are easily distinguishable from the microspheres and can be quantitated on the basis of their light scattering and fluorescence properties is contour plots. As the number of microspheres/sample is known, the relative proportions of cells and microspheres, respectively, can be converted into absolute cell numbers. This quick method is useful for any type of studies where cell numbers less than 1 x 10(5) in a small volume are to be determined.

Animals↗
PubMed1985

A comparison of human breast cancer cell kinetics measured by flow cytometry and thymidine labeling.

Flow cytometric determination of tumor ploidy and S-phase fraction following collagenase dissociation and thymidine labeling was performed on 75 consecutive breast cancers. Estrogen and progesterone receptor levels and routine histologic examination also were obtained on each tumor. Cell viability following collagenase dissociation varied from 13 to 95% with a mean of 71%. Thirty-six tumors were diploid, four tetraploid, and four hypertetraploid, and the remainder had DNA indices between 1.1 and 1.9. There was no significant correlation between tumor ploidy and tumor size or estrogen receptor positivity or negativity. The percentage of cells in S-phase varied from 1.2 to 20.0% with a mean of 6.0% utilizing a rectilinear model for histogram analysis that integrated a 10-contiguous channel sample containing the lowest number of cells in S-phase (S-pFL). The mean S-pFL of diploid carcinomas (3.43%) was significantly lower than that of hyperdiploid carcinomas (8.38%). There was good correlation between S-phase fraction determined by thymidine-labeling index (TLI) and S-pFL (r = 0.772, p = 0.0001). S-pFL predicted whether a tumor would be above or below median TLI with an accuracy of 90.5%. Estrogen receptor-negative cancers tended to have higher TLIs and S-pFLs than estrogen receptor-positive cancers; however, there was no correlation between progesterone receptor positivity or negativity and TLI and S-pFL.

Breast Neoplasms↗
PubMed1986

Flow cytometry in urologic oncology.

Flow cytometers have proved to be useful research tools in cellular biology. By using these devices, rapid and objective measurements of a variety of cellular parameters can be made on a cell-by-cell basis. The application of flow cytometers to the study of urologic neoplasms is at present very preliminary. The available literature on the flow cytometric study of urologic tumors is presented and discussed.

DNA, Neoplasm↗
PubMed1986

[Analysis of the chromosome number in cells by flow cytometry].

A new flow-cytometric method is described that permits analysis of the number of chromosomes of individual cells. Preliminary stained mitotic cells are passed through a specially designed flow chamber in which they are destroyed just before passing through the focused laser beam of the flow cytometer. Signals from chromosomes of the destroyed cells are counted, and the results are represented as a distribution of the number of chromosomes in the cells. The method may be applied for the detection of relatively rare cells with abnormal numbers of chromosomes in biological and clinical research.

Analog-Digital Conversion↗
PubMedDate not supplied

[Analysis of DNA degradation in the thymocytes of irradiated rats by flow cytometry].

The method of flow cytofluorometry of cells treated with probes specifically bound to AT- or GC-pairs of DNA was used to study DNA degradation in thymocytes of irradiated and hydrocortisone-treated rats. Death of thymocytes was shown to be accompanied by the decrease in the DNA content. The main regularities in the formation and accumulation of cells, the DNA content of which being lower than that of diploid cells, were the same as those of the internucleosome DNA fragmentation.

Animals↗
PubMed1994

Differentiation of borreliacidal activity caused by immune serum or antimicrobial agents by flow cytometry.

We demonstrated that borreliacidal activity caused by immune serum and complement can easily be differentiated by flow cytometry from killing activity caused by antimicrobial agents that are commonly used for the treatment of Lyme disease. Assay suspensions containing normal or immune serum were incubated with Borrelia burgdorferi in the presence or absence of ceftriaxone, doxycycline, penicillin, and phosphomycin for 2, 8, 16, and 24 h. Samples containing killing activity were identified by using flow cytometry and acridine orange. In 30 min, the effects of immune serum and complement were easily distinguished from the killing of spirochetes by antimicrobial agents by adding fluorescein isothiocyanate-conjugated goat anti-hamster immunoglobulin. This simple procedure greatly enhanced the usefulness of the borreliacidal assay by eliminating a major source of false-positive reactions.

Acridine Orange↗
PubMed1995

Rapid detection of hereditary and acquired platelet storage pool deficiency by flow cytometry.

Platelet aggregation is commonly used to investigate patients with possible dense granule storage pool deficiency (delta SPD), but recent studies have shown that this investigation is not specific or sensitive for this disorder. We describe a simple one-step technique to detect mepacrine loaded platelets by flow cytometry and found a good correlation (r = 0.83) between this method and the enumeration of platelet dense granules by conventional fluorescent microscopy. Seven patients with congenital delta SPD had significantly (P < 0.001) reduced mepacrine labelling detected by flow cytometry (mean 15%; range 5-23%) compared to normal controls (mean 48%; normal range 32-64%). Six patients with other hereditary platelet disorders had normal mepacrine labelling (mean 49%; range 34-66%) and were clearly distinguished from patients with delta SPD despite similar platelet aggregation patterns. Acquired delta SPD is frequently associated with the platelet function defect described in the myeloproliferative and myelodysplastic disorders (MPD/MDS) and we compared platelet aggregation and mepacrine labelling in 15 of these patients. The results confirm that delta SPD occurs commonly in MPD/MDS with 7/15 patients having reduced mepacrine staining but, like the findings in hereditary delta SPD, 3/7 patients with normal platelet aggregation had delta SPD. Similarly abnormal platelet aggregation was not diagnostic of delta SPD as 4/8 of these patients had normal mepacrine levels. These results may contribute to the known lack of correlation between the limited assessment of platelet function and bleeding events in MPD/MDS. We found mepacrine labelling of platelets detected by flow cytometry to be a useful, simple and inexpensive method to detect hereditary and acquired delta SPD which will improve the definition of the platelet defect in these disorders in the clinical laboratory.

Adult↗
PubMed1996

Flow cytometry analysis of alpha1-adrenoceptor subtypes.

To characterize the alpha1-adrenoceptor subtypes, we developed a flow cytometry method using the fluorescent ligand BODIPY-FL prazosin and the anti-peptide antibody against the alpha1b-adrenoceptor amino terminus (designated 1B-N1-C) as probes. Three alpha1-adrenoceptors (alpha1a, alpha1b and alpha1d) expressed in CHO cells were detected by BODIPY-FL prazosin; however, only alpha1b-adrenoceptor subtype was detected by the anti-peptide antibody 1B-N1-C. Furthermore, the flow cytometry analysis with 1B-N1-C specifically identified alpha1b-adrenoceptor in native cells of hamster DDT1-MF2 cells, rat hepatocytes and cardiomyocytes.

Amino Acid Sequence↗
PubMed2003

Aspirin treatment does not attenuate platelet or leukocyte activation as monitored by whole blood flow cytometry.

Despite undoubtful clinical evidence of anti-platelet effects of aspirin, previous studies demonstrate little inhibition by aspirin treatment of single platelet activation as measured by flow cytometry. This discrepancy was further evaluated using flow cytometric measurements of circulating platelet-leukocyte aggregates (PLAs), which may be a more sensitive marker of platelet activation in vivo than measurements of activation markers on single platelets. Blood samples were obtained from 15 healthy subjects before and after aspirin treatment (75 and 500 mg daily for 1 week). Platelet (P-selectin expression) and leukocyte (CD11b expression) activation and platelet-leukocyte aggregation were monitored by whole blood flow cytometry. Approximately 1% platelets in unstimulated samples were P-selectin-positive, i.e., circulating activated platelets, and about 3% of leukocytes were circulating as PLAs; neither of these parameters were reduced by aspirin treatment. Circulating platelet micro-aggregates were not influenced by aspirin either. In vitro stimulation with ADP, thrombin, or PAF increased platelet P-selectin expression and thus PLA formation, but these responses were not affected by aspirin. Leukocyte CD11b expression, a marker of leukocyte secretion, was not significantly influenced by aspirin either in unstimulated samples or upon in vitro stimulation. Thus, the present data support the concept that thromboxane generation is of little importance for the activation of single platelets; the platelet inhibiting effect of aspirin is seen mainly in the presence of close cell-cell contact, which enhances the importance of thromboxane. Multiple mechanisms may contribute to the remarkable clinical anti-thrombotic effect of aspirin.

Adult↗
PubMed1999

Flow cytometry: a versatile tool for all phases of drug discovery.

The applications of flow cytometry are being extended beyond cells into molecular interactions and genomic analyses. The authors explain how instrumentation and reagent development are combining to improve flow cytometric throughput by orders of magnitude beyond that possible just a few years ago, such that the combination of sensitivity, throughput and versatility makes flow cytometry an analytical platform with applications at all stages of drug discovery.

Journal Article↗
PubMed1999

Tandem application of flow cytometry and polymerase chain reaction for comprehensive detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Children with acute lymphoblastic leukemia (ALL) with > or = 0.01% leukemic cells in the bone marrow after remission induction are at a greater risk of relapse. The most promising methods of detecting minimal residual disease (MRD) are flow cytometric identification of leukemia-associated immunophenotypes and polymerase chain reaction (PCR) amplification of antigen-receptor genes. However, neither assay can be applied to all patients. Moreover, both assays carry the risk of false-negative findings due to clonal evolution. The simultaneous use of both assays might resolve these problems, but the correlation between the methods is unknown. We studied serial dilutions of normal and leukemic cells by flow cytometry and PCR amplification of IgH genes and found the two methods highly sensitive (one leukemic cell among 10(4) or more normal cells), accurate (r2 was 0.999 for flow cytometry and 0.960 for PCR by regression analysis) and concordant (r2 = 0.962). We then examined 62 bone marrow samples collected from children with ALL in clinical remission. In 12 samples, both techniques detected MRD levels > or = 1 in 10(4). The percentages of leukemic cells measured by the two methods correlated well (r2 = 0.978). Of the remaining 50 samples, 48 had MRD levels < 1 in 10(4). In only two samples results were discordant: 2 in 10(4) and 5 in 10(4) leukemic cells by PCR but < 1 in 10(4) by flow cytometry. We conclude that immunologic and molecular techniques can be used in tandem for universal monitoring of MRD in childhood ALL.

Child↗
PubMed1998

Viability assessment of dog spermatozoa using flow cytometry.

The percentages of living and dead spermatozoa in fresh dog semen samples were assessed by means of a dual staining technique using carboxifluorescein diacetate (CFDA) and propidium iodide (PI). Two ejaculates were obtained from dogs, each ejaculate was divided into 4 aliquots, and different proportions of freeze-killed cells were added to each aliquot. Data obtained by flow cytometry analysis of each sample were compared with those obtained by the microscopic evaluation under epifluorescence illumination and by phase-contrast microscopy evaluation of the samples stained with eosin-nigrosin. Regression analysis was used to compare the 3 methods for membrane integrity assessment of canine spermatozoa, and high correlation coefficients were found between the flow cytometry procedure and the 2 microscopy techniques. The results from this study validate the use of flow cytometry as a precise method for assessing the viability of dog spermatozoa.

Animals↗
PubMed2002

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay.

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.

Annexin A5↗
PubMed1996

Differential expression of T cell antigens in normal peripheral blood lymphocytes: a quantitative analysis by flow cytometry.

AIMS: To obtain reference values of the level of expression of T cell antigens on normal lymphocyte subsets in order to disclose differences which could reflect their function or maturation stages, or both. METHODS: Peripheral blood from 15 healthy donors was processed by flow cytometry with triple colour analysis. For each sample phycoerythrin (PE) conjugated CD2, CD4, CD5, CD8, and CD56 monoclonal antibodies were combined with Cy5-R-phycoerythrin (TC) conjugated CD3 and fluorescein isothiocyanate (FITC) conjugated CD7; CD2- and CD7-PE were also combined with CD3-TC and CD4-FITC. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values of the lymphocyte subsets identified by multiparametric flow cytometry into the number of antigen molecules per cell, measured as antibody binding capacity (ABC). RESULTS: CD4+ (helper/inducer) T cells exhibit a higher CD3 antigen expression compared with CD8+ (suppressor/ cytotoxic) T lymphocytes. Within the CD4+ T cells, the CD4+CD7- subset expressed a lower level of CD3 compared with CD4+CD7+ and CD8+CD7+ cells, and higher CD2 and CD5 expression than the main CD3+CD7+ subset. Major differences in antigen expression were also detected between CD3+ T cells and CD3-CD56+ natural killer (NK) cells: NK cells exhibited higher levels of CD7 and CD56 and lower levels of CD2 and CD5 than T cells. Significantly lower CD5 expression was also detected in the small CD5+ B lymphocyte subset compared with T cells. CONCLUSIONS: Quantitative flow cytometry with triple colour analysis may be used to detect antigen modulations in disease states and to increase the accuracy of diagnosis by comparison with findings in normal counterparts.

Adult↗
PubMed2007

Thematic workshop on fluorescence compensation settings in multicolor flow cytometry.

In his program of thematic one-day workshops, the French Association of Cytometry had organized a workshop dedicated to the fluorescence compensation settings in multicolor flow cytometry. This special day was in honor of our past President Jean Luc D'Hautcourt who has been involved in the quality of the use of flow cytometry in its clinical and research purposes. Review on fluorescence phenomena, compensation rules, settings, and few observed confounding situations were presented.

CD4-Positive T-Lymphocytes↗
PubMed1992

Detection of antigen specific T lymphocytes by determination of intracellular calcium concentration using flow cytometry.

We present a method for the detection of lymphocytes with specific reactivity to antigens on stimulator cells using flow cytometry. Cultured human T lymphocytes were loaded with the intracellular fluorochrome indo-1 and were mixed with stimulator cells. Using flow cytometry we could detect a specific increase in intracellular calcium in the T lymphocytes as well as conjugation between the T cells and the stimulator cells. Examination of antigen-specific CD4+ and CD8+ T cell clones demonstrated that the vast majority of T cells which were conjugated to antigen-bearing stimulator cells manifested a rapid increase in intracellular calcium. In contrast T cells conjugated to stimulator cells which did not bear specific antigen demonstrated no such increase in calcium. A similar finding was observed when examining polyclonal tumor infiltrating lymphocytes obtained from patients with melanoma. Tumor infiltrating lymphocytes with specific antitumor reactivity demonstrated an increase in intracellular calcium when conjugated to autologous tumor but not to allogeneic melanoma. In contrast to the T cell clones, only a small subpopulation of tumor infiltrating lymphocytes manifested this specific signal upon conjugation with autologous tumor. This suggests that tumor infiltrating lymphocyte cultures contain T cells with varying reactivities to tumor or may also imply heterogeneity in the stimulating tumor cell lines. The method allows for the detection of specific T cells on an individual cell basis in real time. The procedure is not lethal to the cell and sorting and subculturing of reactive T cell populations can be readily performed. The method could also be used to sort stimulator cells based on their ability to elicit an increase in intracellular calcium in selected T cells.

CD4 Antigens↗
PubMed1992

Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry.

We have developed a technique for analysis of granulocyte reactive oxygen species formation in whole blood using flow cytometry and two color immunofluorescence. This technique relies upon the use of specific fluorescent dye (LDS-751) to stain nucleated cells, eliminating erythrocytes from analysis. Using LDS-751, forward angle light scatter, and 90 degrees side scatter, a granulocyte gate, monocyte gate, and lymphocyte gate were identified. Analysis with multiple FITC conjugated monoclonal antibodies demonstrated greater than 95% purity of a flow cytometrically identified granulocyte population in whole blood without physical manipulation of the blood. Utilizing 2'7' dichlorofluorescein diacetate (DCFH-DA), we were able to measure granulocyte intracellular reactive oxygen species production. Dose response curves were obtained for the effect of granulocyte agonists phorbol myristate acetate, FMLP, and heat fixed Staphylococcus aureus on reactive oxygen species production. The techniques described in this paper should be useful for measuring granulocyte activation in vivo with flow cytometry.

Cell Separation↗