PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Flow Cytometry”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed2000

Detection of dead cells and measurement of cell killing by flow cytometry.

A flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell within a large, heterogeneous population. The modern commercially available analytical instruments, which can be found in most hospitals, pathology laboratories, and cell biology research laboratories in the industrially developed countries, can now routinely measure fluorescence simultaneously at four different wavelengths, in addition to light scatter in two directions, at rates of thousands of cells per second. Thus, flow cytometry provides a powerful and versatile approach to the measurement of cell death and cell killing. However, it is important to stress that the biological nature of the phenomenon under investigation will determine which assay is appropriate, and at what stage in the process of cell killing it should be applied. This consideration is particularly important in the study of drug- or cell-mediated cytotoxicity.

Animals↗
PubMed1992

Relationship between sperm quality and chromatin condensation measured by sperm DNA fluorescence using flow cytometry.

DNA flow cytometry of sperm from 100 randomly chosen men undergoing fertility investigation revealed a general association between reduced sperm quality, as judged by conventional parameters, and the appearance of sperm with lower degrees of chromatin condensation in the ejaculate as measured by DNA fluorescence intensity. Chromatin hypocondensation, as measured by increased fluorescence, was manifested to different degrees in different samples. In many cases of more extreme sperm pathology, such as oligoasthenoteratozoospermia (OAT), the whole population of spermatozoa appeared to be affected. Significant numbers of hypercondensed spermatozoa were present in both normozoospermic men and men with different degrees of disturbance in sperm quality. All of the different parameters of sperm quality could be correlated significantly with certain of the flow parameters, although not one in particular could be used to predict deviations from the normal flow profile. In several asthenoteratozoospermic men and a small proportion of men with OAT, the DNA profiles were normal, implying that in these cases the disturbance may not be so fundamental. The presence of leucocytes in the ejaculate was associated with a general increase in the preponderance of hypocondensed subpopulations of spermatozoa in men with OAT as well as in normozoospermic subjects, emphasizing the effect of inflammatory conditions in the reproductive tract on sperm quality.

Chi-Square Distribution↗
PubMed1989

Lymphocyte immunophenotyping in an elderly population: age, sex and medication effects--a flow cytometry study.

Flow cytometry studies of 179 individuals aged 55-95 years reveal correlations of various hematological and immune cell measures (quantification of leukocytes, leukocyte subtypes, platelets, and erythrocytes) with age, sex, common medications (aspirin, ulcer therapy, estrogen replacement therapy), and health history variables (minor arthritis, allergy, smoking, stress and exercise). Possible reasons for some of these findings are discussed.

Age Factors↗
PubMed1996

Competence effect of basic fibroblast growth factor on cell cycle in subcultured endothelial cells of rat aorta by flow cytometry.

With flow cytometry, we investigated the effect of basic fibroblast growth factor (bFGF)-induced competence in subcultured endothelial cells (EC) (4-9-passage) of rat thoracic aorta. The cell population in each phase of the cell cycle was determined by a double staining technique with fluorescein isothiocyanate-conjugated mouse monoclonal antibody against the proliferation-associated nucleus antigen Ki-67 and propidium iodide for total DNA content. EC were cultured in medium containing 5% fetal bovine serum (FBS) for 6 days. After serum-starvation for 2 days, the treatment with bFGF (3-10 ng/ml) for 12 hr promoted the entry of cells into the G1 phase from the G0 phase concentration-dependently. bFGF (10 ng/ml) increased the cell population in the G1 phase by 5% of the total EC, compared with the control culture without bFGF. A further 12-15-hr culture with 1% FBS after bFGF treatment promoted the entry of the cell into the S phase. Thus flow cytometric analysis demonstrates that bFGF stimulates the entry of EC into the G1 phase from the G0 phase.

Animals↗
PubMed1988

Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry.

A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.

Antibodies↗
PubMed1992

[DNA synthesis and content in human decidual cells at different stages of differentiation based on flow cytometry data].

Flow cytometry analysis of the DNA content of human decidual cells at the physiologically normal pregnancy and in the case of toxicosis was carried out. During the normal pregnancy DNA-histogram parameters were seen to vary: the number of S-phase cells decreased, the coefficient of variation of the G1/0 peak increased. These alterations correlated positively with the increase in the share of decidual cells with properties of terminally differentiated cells. The most pronounced quantitative alterations in variability of DNA content in G1/0 cells were observed in cases of toxicosis of pregnancy. Phenomena of variability of the nuclear DNA in the terminally differentiated decidual cells is considered to be a sign of cell death through apoptosis.

Adolescent↗
PubMed2006

A simple method for quantifying high density antigens in erythrocyte membrane by flow cytometry.

RBC flow cytometric analysis is usually used to quantify antigen content. Calibration systems enable antigen content determination by relating mean fluorescence intensity with the number of bound antibody molecules (equivalent to the number of antigen molecules). For that reason, antibodies must be used at saturating concentration, which may lead to agglutination when working with high density antigens. Then, forward scattering, side scattering and fluorescence will be increased, thus obtaining wrong results. In this work, the simple Langmuir adhesion model was applied. Flow cytometry was used to quantify GPA, a transmembrane protein present at high density on RBC. The fluorescence intensity of samples at different anti-GPA sub-saturating concentrations was measured. Sometimes, agglutinates were present and two peaks of fluorescence were observed, the principal one corresponding to isolated cells and the secondary one corresponding to agglutinated cells. In those cases, the principal peak was taken into account for the analysis. The GPA antigen content obtained for nine analyzed samples ranged from 3 to 13 x 10(5) sites per cell, which is similar to those values found in literature. Therefore, the Langmuir adsorption model enables us to determine the antigen content for the anti-GPA/GPA system on RBC membrane. This model could be used to quantify high density antigens in RBC and in other cells.

Antibodies, Monoclonal↗
PubMed1997

Separation of cells at different times within G2 and mitosis by cyclin B1 flow cytometry.

Multivariate flow cytometry using specific cyclin proteins and DNA content can identify cell populations at different points within the cell cycle. Quantification of cyclin B1 and DNA content reveals that cells with high levels of cyclin B1 predominantly have a 4C DNA content and are therefore in G2 or mitosis. We have examined whether separation of cells by levels of cyclin B1 could be used to discriminate cells at discrete times within these phases. Post-replicative cells progressively enter into fractions with higher levels of cyclin B1, indicating that this protein can be used as a marker of time in G2. Furthermore, cells in particular phases of mitosis can be greatly enriched by separation based on cyclin B1 levels. This method can thus be used to isolate cells at specific times within G2 and mitosis, periods of the cell cycle that have been difficult to study by cell fractionation.

Adult↗
PubMed1992

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

Fluorescence flow cytometry was employed to assess the potential of a vital dye, hydroethidine, for use in the detection and monitoring of the viability of hemoparasites in infected erythrocytes, using Babesia bovis as a model parasite. The studies demonstrated that hydroethidine is taken up by B. bovis and metabolically converted to the DNA binding fluorochrome, ethidium. Following uptake of the dye, erythrocytes containing viable parasites were readily distinguished and quantitated. Timed studies with the parasiticidal drug, Ganaseg, showed that it is possible to use the fluorochrome assay to monitor the effects of the drug on the rate of replication and viability of B. bovis in culture. The assay provides a rapid method for evaluation of the in vitro effect of drugs on hemoparasites and for analysis of the effect of various components of the immune response, such as lymphokines, monocyte products, antibodies, and effector cells (T, NK, LAK, ADCC) on the growth and viability of intraerythrocytic parasites.

Animals↗
PubMed1999

The age-dependent cytokine production by murine CD8+ T cells as determined by four-color flow cytometry analysis.

Flow cytometry has been used to simultaneously examine intracellular cytokine production and expression of CD44 and CD45RB by murine CD8+ T cells derived from young (2-3 months) or aged (18-22 months) mice. Cytokine production by aged CD8+ T cells differs from that by CD8+ T cells derived from young animals in that a significantly higher percentage of the aged can be triggered to produce interleukin (IL)-4, interferon (IFN)-gamma, and tumor necrosis factor alpha (TNF alpha). Conversely, a greater fraction of young CD8+ T cells produce IL-2. Aged mice not only have a higher percentage of CD8+/CD44hi T cells, but also a larger fraction of these cells are IFN-gamma+ and IL-4+, while a lower fraction are IL-2+ than is observed in young CD8+/CD44hi T cells. In terms of relative contribution to total cytokine synthesis, a greater fraction of CD8+ T cells produce IFN-gamma and IL-4 than in CD4+ T cells, whereas CD4+ T cells are the major producers of IL-2.

Aging↗
PubMed1996

Dynamics of donor-reactive IgG, IgA and IgM antibodies against T and B lymphocytes early after clinical kidney transplantation using flow cytometry.

Using flow cytometry, 32 kidney graft recipients were monitored retrospectively for at least 1 month to study the dynamics of serum IgG, IgA and IgM antibodies against donor T and B lymphocytes before and after transplantation. Donor spleen lymphocytes were used as targets. In the B cell cross-match, the surface immunoglobulins were blocked with an anti-human immunoglobulin antibody. A high frequency of donor-reactive antibodies was detected early after transplantation, especially when the sera were tested against B lymphocytes. Surprisingly, donor-reactive antibodies of the IgA isotype made up a substantial proportion of all antibodies detected. Within the first month after transplantation, six out of 32 patients (19%) developed IgG antibodies against donor T lymphocytes and nine out of 35 patients (28%) formed IgG antibodies against B lymphocytes. A similar situation was found for IgA antibodies: 22% of the recipients were positive for IgA antibodies against donor T lymphocytes and 34% against B lymphocytes after transplantation. Lower antibody frequencies were found for IgM antibodies (16% and 19%, respectively). From our data we conclude that for at least some of the parameters studied their individual dynamics reflect the complex immunological mechanisms occurring early after transplantation.

Antibodies, Anti-Idiotypic↗
PubMed1987

A single laser method for subtraction of cell autofluorescence in flow cytometry.

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.

Animals↗
PubMed1994

Detection of respiratory syncytial virus infection in nasal aspirate samples by flow cytometry.

HYPOTHESES: (i) Flow cytometry has the potential for rapid detection of respiratory viral antigens. (ii) This technique can be applied to viral diagnosis in clinical samples. OBJECTIVES AND STUDY DESIGN: (i) To study the identification of six common respiratory viral pathogens by flow cytometry, in virus infected and uninfected cultured cells, as models of positive and negative clinical samples. (ii) To compare flow cytometry with the established techniques of viral isolation and immunofluorescent microscopy in the diagnosis of respiratory syncytial virus infection in 68 naso-pharyngeal aspirates taken from children and sent to the virology laboratory for routine virological diagnosis. RESULTS: (i) For each virus analysed, populations of infected and non-infected cells were clearly discernable, confirming potential for this method in rapid viral diagnosis in clinical samples. (ii) Two definitions were employed for a sample to be positive by flow cytometry, these were compared with the combined established techniques. The sensitivity, specificity, positive and negative predictive values of flow cytometry were 41%, 98%, 92% and 71% for the first definition and 74%, 88%, 80% and 84% for the second definition respectively. CONCLUSIONS: As tested in this study, flow cytometry is less sensitive than established techniques as well as recently developed rapid diagnostic techniques for the diagnosis of respiratory syncytial virus infection. Further evaluation of the potential of flow cytometry in rapid viral diagnosis is warranted.

Journal Article↗
PubMed1992

Detection of antibiotic-induced platelet dysfunction in whole blood using flow cytometry.

Using flow cytometry and activation-dependent monoclonal antibodies, we have developed a technique based on forward angle-light scatter (FALS) and immunofluorescence that simultaneously detects human platelet activation, secretion, and aggregation in whole blood. To detect the effects of cefotetan and latamoxef, both of which contain an N-MTT side chain, and of free N-MTT and cefoxitin, which does not contain the N-MTT side chain, on platelet activation and secretion, platelets were stained by the indirect method using a murine-produced platelet specific activation-dependent monoclonal antibody, S12, and a goat anti-mouse IgG fluorescein-conjugated antibody. S12 binds to a 140kd alpha granule membrane protein (GMP-140) that is expressed during secretion. Single parameter, 256 channel, log integrated green fluorescence histograms were generated, and negative and positive fluorescent populations were defined. Latamoxef and cefotetan reduced the number of platelets expressing S12 by more than 43%. In contrast, cefoxitin reduced the number of platelets expressing S12 by only 13.5%. The inhibition of GMP-140 expression per platelet was calculated by converting the log data to linear fluorescence intensity. Latamoxef and cefotetan inhibited expression of GMP-140 by 88% and 87% respectively. Free N-MTT inhibited its expression by 68%. In contrast cefoxitin reduced GMP-140 expression per platelet by only 45%.

Adenosine Diphosphate↗
PubMedDate not supplied

Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry.

A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells.

Animals↗
PubMed1994

Intratumoral DNA heterogeneity correlated with lymph node involvement and surgical staging in epithelial ovarian cancer by flow cytometry.

BACKGROUND: Flow cytometry (FCM)-measured DNA content may be a predictor in the prognosis of ovarian cancer. Multiple specimens taken from the same ovarian tumor may show a variation in DNA content (i.e., intratumoral DNA heterogeneity). We measured the FCM DNA content of multiple specimens from the same tumor in ovarian cancer, and the relationship among DNA ploidy, intratumoral DNA heterogeneity, retroperitoneal lymph node involvement, and surgical staging was evaluated. METHODS: Forty-one patients with primary epithelial ovarian cancer were included in the study. The FCM-measured DNA content of multiple fresh tumor specimens taken from different parts of the same ovarian tumor from each patient was measured. When aneuploidy was observed in at least one specimen from the same tumor, the tumor was defined as an aneuploid tumor. If there were two or more different aneuploid stem lines with a variation of DNA indices (differences of the DNA indices > 0.15) from the same tumor, the presence of intratumoral DNA heterogeneity was defined. RESULTS: Diploid tumor was found in 8 (19.5%) of the 41 patients, and aneuploid tumor with intratumoral DNA heterogeneity was found in 20 (48.8%). None of the eight patients with diploid tumors demonstrated lymph node involvement. In contrast, lymph node involvement was found in 14 (70.0%) of 20 patients with intratumoral DNA heterogeneity. There was a significant different incidence of lymph node involvement between the groups with and without intratumoral DNA heterogeneity (P < 0.01). The incidence of intratumoral DNA heterogeneity significantly correlated with the International Federation of Gynecology and Obstetrics staging (P < 0.01), while that of aneuploid tumor did not. CONCLUSIONS: Intratumoral DNA heterogeneity reflected a malignant potential for lymph node involvement and its progression in epithelial ovarian cancer.

Aneuploidy↗
PubMed1999

Rapid detection of Salmonella in chicken washes by immunomagnetic separation and flow cytometry.

Use of flow cytometry to rapidly detect Salmonella in chicken carcass washes was investigated. A direct immunomagnetic separation method was used to prepare samples and was found to be an effective method for separating target cells from debris in chicken carcass washes. When flow cytometry was combined with immunomagnetic separation, the average lowest detectable level of Salmonella detected was 2.3 x 10(4) CFU/ml. Fifty of 100 wash samples from six groups were inoculated with 2 x 10(-1) CFU of Salmonella Typhimurium per milliliter. After 18 h of enrichment at 37 degrees C, all samples were tested for Salmonella using flow cytometry and conventional culture methods. An identification correlation of 96% was found between flow cytometry and xylose-lysine-tergitol agar plating. Quantitative analysis established a significant linear relationship between the enumeration results of flow cytometry and xylose-lysine-tergitol agar plate counts (R2 = 0.796). Time required for flow cytometry, including sample processing and flow cytometric analysis, was less than 1 h.

Animals↗