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Results for “Flow Cytometry”

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PubMed1996

[Analysis of surface antigen molecule expression on serovar stains of Neisseria gonorrhoeae by flow cytometry].

In the present experiment, flow cytometry was employed for analysing expression characteristics of the antigen molecules distingushed by 10 monoclonal antibodies against Neisseria gonorrhoeae lipooligosaccharides. Stability of antigen expression and amount of epitope on the surface of N. gonorrhoeae were quantitative determined. Reactivity of the monoclonal antibodies with serovar strains of N. gonorrhoeae were evaluated.

Antibodies, Monoclonal↗

PubMed1994

Dating the endometrial biopsy by flow cytometry.

OBJECTIVE: To determine endometrial changes throughout the menstrual cycle. DESIGN: Flow cytometric analysis of endometrial cells versus chronological dating. SETTING: Women volunteers with a normal menstrual cycle participated in this study that was done in an academic research environment. PATIENTS: Two hundred thirty regular menstruating women with adequate luteal phase underwent endometrial biopsy at different days of their menstrual cycle; 138 biopsies were analyzed both histologically and by flow cytometry. MAIN OUTCOME MEASURE: Percentage of biopsies correctly classified in terms of chronological dating. RESULTS: Flow cytometry correctly classified the chronological day of biopsies in 59% of cases, the proliferative versus secretory days in 91% of cases, and the early, mid, and late secretory phases of the menstrual cycle were correctly predicted in 86% of cases. CONCLUSION: Flow cytometry is a good method to determine growth and differentiation of the endometrium during the menstrual cycle. Detection of phases of the whole endometrial cell population offer information on the biological adequacy of endometrial response to the biochemical environment.

Biopsy↗

PubMed2006

Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry.

The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.

Bioreactors↗

PubMed1997

A simultaneous negative and positive selection method that can detect chimerism at a frequency of 1 per 10,000 by flow cytometry.

A three-color flow cytometric method with a simultaneous negative and positive selection (SNAPS) approach has been developed to detect blood chimerism using a FITC-conjugated recipient-specific HLA monoclonal antibody, a biotinylated donor-specific HLA monoclonal antibody and Cy5-PE-conjugated streptavidin and a PE-conjugated CD3 monoclonal antibody. Detection of simulated blood chimerism at a frequency of 0.01% was achieved by SNAPS. In contrast, by one-color flow cytometry methods, chimerism can be detected at only about 3-4% with the same donor-specific HLA monoclonal antibody. The percentages of donor CD3 cells detected by SNAPS are proportional to the percentages of donor CD3 cells counted. SNAPS method provides a fast, sensitive and reliable way to analyze trace amounts of donor cells in a recipient's blood.

Animals↗

PubMed1984

Flow cytometry of the urinary bladder.

Flow cytometry now appears sufficiently developed technically to identify carcinoma of the bladder on irrigation specimens with accuracy comparable to that of conventional exfoliative cytology. The cancer cells are distinguished by abnormal levels of DNA per cell, which can be quantified with acridine orange, a metachromatic fluorescent dye for DNA and RNA. With this technique it is also possible to follow the development of carcinoma and assess the effects of treatment by quantifying the number of cancer cells present in the irrigation specimen.

BCG Vaccine↗

PubMed2000

A standardized gating technique for the generation of flow cytometry data for normal canine and normal feline blood lymphocytes.

Flow cytometry is becoming a commonly used technique to characterize a variety of cells. It provides a powerful application to rapidly determine the relative percentages of T-lymphocyte subsets and B-lymphocytes. The effectiveness of its application, however, is dependent on standardization, especially in a clinical setting. Application of flow cytometry to veterinary diagnostics has been limited by the unavailability of reagents and by the unstandardized characterization of normal values using antibodies not commercially available, but typically provided through the generosity of other researchers. This paper presents a standardized gating protocol, and average values and ranges observed for normal canine and feline blood lymphocytes using commercially available antibodies to cell surface markers for CD5, CD3, CD4, CD8, MHC II, and B lymphocytes. The averages for these markers on gated lymphocytes were as follows: Canine CD5 83.3%, Canine CD4 45.0%, Canine CD8 28.8%, Canine MHC II 98.0%, Canine B Cell 12.9%, Canine CD4/CD8 ratio 1.87, Feline T lymphocytes 77.3%, Feline CD4 44.5%, Feline CD8 25.7%, Feline B Cell 24.1%, Feline CD4/CD8 Ratio 1.75. Normal values were also established for a mixed breed group of dogs, and old versus young dogs. This information will provide researchers and clinicians with a standardized protocol for gating, which establishes a basis for comparison between techniques, and a measure of phenotypic percentages for flow cytometry in normal dogs and cats based on this standardization and commercially available antibodies.

Age Factors↗

PubMedDate not supplied

Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques.

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.

Animals↗

PubMed1984

Purification of beta cells from rat islets by monoclonal antibody-fluorescence flow cytometry.

Fluoresceinated monoclonal antibody plus flow cytometry was used to purify beta cells from mixed pancreatic islet endocrine cell populations. A2B5, a monoclonal antibody to a glycolipid on the surface of cells of neuroendocrine origin, was incubated with single cells dissociated from rat pancreatic islets. Antibody-bound cells were labeled with fluoresceinated goat F(ab')2 antimouse immunoglobulin and highly fluorescent cells were separated from less fluorescent cells on a Coulter EPICS IV cell sorter. Sorted cell populations were analyzed by radioimmunoassay for insulin, glucagon, and somatostatin. The highly fluorescent cell population was enriched sixfold for insulin-containing beta cells, indicating that islet beta cells are relatively enriched in A2B5 antigen and can be partially purified by this method.

Animals↗

PubMed1992

Application of flow cytometry in clinical renal transplantation.

Flow cytometry (FC) may be considered as a fundamental technique in studying cell biology and pathology. It combines the quantitative character of biochemical methods with the multiparametric capacities of microscope analysis in a high-precision process for rapid analysis of individual cell characteristics. Three original FC techniques routinely applied in the field of renal transplantation are reported in the present study. They concern the donor-recipient cross-match test, the morphological analysis of urinary sediment and the modulation of the density of various membrane antigens on the lymphocyte surface. A common factor underlies all these methods: they aim to provide the physician with a reliable diagnostic tool in clinical renal transplantation.

Antigens, Surface↗

PubMed1981

Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry.

Phagocytic particle uptake was calculated from data obtained by microscopy and flow cytometry utilizing commercially available fluorescent beads of uniform (2 micrometer diameter) size. PMNs collected from a Ficoll-Hypaque density gradient were incubated with the fluorescent beads in human plasma and gently washed in buffer to remove most of the extracellular beads. The cells were observed and analyzed by fluorescence microscopy (sample size = 300 cells) and flow cytometry (sample size = 50,000 cells) to determine the percent phagocytic cells, number of beads per phagocytic cell, and beads per 100 PMNs. Correlation of these indices for microscopic and machine counts was in high agreement, with r values greater than 0.80. Since flow cytometry increases the sample size 500-fold above that attained by manual microscopic counts, the coefficient of variation is proportionately reduced. Unlike other automated techniques for phagocytosis such as the use of radiolabeled particles, flow cytometry yields information on the cell population such as percent of phagocytic cells and the distribution of intracellular fluorescent beads per cell as well as particle uptake. Thus the advantages of speed, greater statistical accuracy, and the ability to accumulate information on the cell population indicate that flow cytofluorometry has unique applications for the study of phagocytosis. Application of the technique to in vitro stimulation of blood neutrophil phagocytic uptake is included.

Cytological Techniques↗

PubMed1982

Flow cytometry of human testicular tissue.

Flow cytometry of testicular tissue from autopsies showed differences in the proportion of substained spermatozoa (1 CA), spermatids (1 CB), diploid 2C nuclei and premeiotic spermatozoa in the 4 C class. The evaluation of the histograms was performed with computer assistance. Healthy accident cases or those delivered in forensic medicine furnished the control values. Patients with hepatic cirrhosis showed a nearly complete loss of the haploid class, while cases with fatty degeneration of the liver demonstrated a reduction. Circulatory insufficiency in cases of acute shock resulted in an increase in the 4C values of meiotic prophase I and a decrease of spermatids. Spermatozoa were normal in these cases. After prolonged shock a nearly complete breakdown of spermatogenesis was observed. In cases of atherosclerosis an irregular degree of reduction was seen. Cancer patients showed a reduction of spermatogenesis in correlation to therapy.

Adult↗

PubMed1986

Cell kinetics of normal and perturbed populations measured by incorporation of bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BrdU) incorporation and flow cytometry have been used to measure the kinetics of V79 cells growing at different temperatures in vitro and cells of the murine Sa F tumour growing in vivo. By simultaneously measuring total DNA content and BrdU incorporation in individual cells at different times after pulse labelling with BrdU, it is a simple procedure to quantify the movement of cells through the cell cycle. The method has the advantage of speed (2 X 10(4) or more cells are analysed within a day or so of the experiment) and the ability to analyse the results in different ways. A G2 block in tumour cell progression in vivo is readily detected after a dose as low as 2 Gy. The neutron relative biological effectiveness for this G2 block is probably larger than that for tumour growth delay.

Animals↗

PubMed2000

Flow cytometry for quantification of retrogradely labeled retinal ganglion cells by Fluoro-Gold.

PURPOSE: To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. METHODS: Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. RESULTS: Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 +/- 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. CONCLUSIONS: The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.

Animals↗

PubMed1998

DNA ploidy and cell-cycle analysis in intracranial meningiomas and hemangiopericytomas: a study with high-resolution DNA flow cytometry.

Although various DNA flow-cytometric studies have been performed on meningiomas, the role of DNA ploidy and the S-phase fraction (SPF) in predicting biological tumor behavior remains unresolved. Discrepant results in earlier studies might be due to different preparing, staining and measuring techniques; different quality standards; and lack of sophisticated computer software. In this study, high-resolution DNA flow cytometry using the DNA-specific dye DAPI (4', 6'-diamidino-2-phenylindol) was performed on stored frozen tissue from 128 microsurgically resected meningiomas and 7 hemangiopericytomas, including 17 recurrent meningiomas and 4 recurrent hemangiopericytomas. The computer software Multicycle 2.5 was used to determine the ploidy level and to perform cell-cycle analysis. DNA aneuploidy and SPF were significantly higher in atypical, anaplastic and recurrent meningiomas and correlated well with histopathological features such as focal necrosis, infiltration of dura mater and mitotic activity. Among 128 meningiomas, 42 had additional DNA aneuploid stem lines. No association between hypo- and hyperploidy and either histological subtype or clinical outcome was found. In 7 hemangiopericytomas, SPF was significantly higher compared to the benign meningioma group, while only 1 tumor was aneuploid. In all 42 DNA aneuploid tumors, cell-cycle analysis was performed separately for the euploid and aneuploid stem lines. The proliferation parameters (SPF, G2/M phase) were significantly higher in the DNA aneuploid stem lines. DNA ploidy and SPF are thus useful indicators of different biological behavior within identical histological subgroups in meningiomas.

Aneuploidy↗

PubMed2002

Control of extracellular matrix homeostasis of normal cartilage by a TGFbeta autocrine pathway. Validation of flow cytometry as a tool to study chondrocyte metabolism in vitro.

OBJECTIVE: To validate flow cytometry as an experimental technique for the study of the homeostasis of the extracellular matrix (ECM) of human articular cartilage. METHODS: Given the established insights in the relation between the transforming growth factor (TGF)-beta type II Receptor (TGF-betaRII)/TGF-beta auto/paracrine pathway, the intracellular levels of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), and the accumulation of ECM molecules in the ECM of articular cartilage, this metabolic pathway was used as a reference model to fulfill the objective. Chondrocytes were liberated from visually intact femoral condyle cartilage and cultured in gelled agarose to maintain their differentiated phenotype. After 2 weeks of culture, the chondrocytes were isolated from the agarose and flow cytometry was used to analyse the expression of TGF-betaRII on the plasmamembrane, the expression of TGFbeta1, MMP-1, MMP-3, TIMP-1 and TIMP-3 inside the cells, as well as the amounts of aggrecan, type II collagen and hyaluronan in the cell-associated matrix (CAM). The expression of the different substances was analysed with flow cytometry and reported as mean fluorescence intensity (MFI), which is due to the binding of FITC-labeled antibodies to their specific antigens. In addition, the effects of exogenous TGFbeta1 on the expression of these proteins was investigated on chondrocytes cultured in serum-free media. Enzyme Linked Immunosorbent Assay (ELISA) was performed to evaluate the MMP-1, MMP-3, TIMP-1 and MMP-1/TIMP-1 complex in the culture medium collected after the last 3 days of the culture period. The correlations between the data were analysed with the Spearman's test. RESULTS: Exogenous TGF-beta1 increased the accumulation of aggrecan and hyaluronan in the CAM of chondrocytes and down-regulated the intracellular levels of MMP-1 and -3. TIMP-1 and -3 were increased after exposure to TGF-beta1. Baseline expression of TGF-betaRII on the plasmamembrane of normal human articular chondrocytes significantly correlated with the intracellular levels of TGFbeta1, TIMP-1 and TIMP-3. TGFbeta1 was correlated with TIMP-1, TIMP-3 and MMP-1. Aggrecan in the CAM was inversely correlated with the ratio of MMP-1 to TIMPs. In addition, there were correlations between TIMP-1 and TIMP-3, aggrecan and hyaluronan. ELISA also revealed the correlation between MMP-1 and TIMP-1 secreted by the chondrocytes into the nutrient medium. MMP-1/TIMP-1 complex was hardly found in the medium. CONCLUSIONS: Some aspects of ECM metabolism of normal cartilage were evaluated by flow cytometry. Chondrocytes from normal human cartilage, when cultured in gelled agarose, showed correlations between the expression of TGF-betaRII/TGF-beta1 and the intracellular levels of TIMPs, indicating that TGF-beta autocrine pathway may contribute to homeostasis of the ECM in the normal cartilage. The relations between MMPs, TIMPs and the ECM molecules support that a physiological balance between MMPs and TIMPs results in a well-controlled matrix turnover in normal cartilage.

Aggrecans↗

PubMed1990

Study of rat renal brush border membrane vesicles by flow cytometry.

The rich heterogeneity of renal tubular membranes and cells continues to provide formidable challenges in the isolation of homogeneous membrane vesicle populations for study. The present study applies flow cytometry, the technique of fluorescence-activated cell sorting, to the study of brush border membrane vesicles. Direct comparison was made of enzymatic marker purity of rat renal cortical brush border membrane vesicles prepared by divalent ion precipitation, or flow cytometry sorting. Flow cytometry sorted membrane vesicles were characterized by greater brush border membrane markers, no detectable mitochondrial or basolateral markers, and greatly reduced Golgi and lysosomal markers. The flow sorted membrane vesicles were functional for transport studies as they took up at least as much 3H-proline and 3H-glucose per mg protein as divalent ion precipitation membrane vesicles. Preparation of membrane vesicles from superficial and deep cortex allowed us to image the different distributions of gamma-glutamyl transferase in membrane vesicles from these areas. Hence, membrane vesicle populations of exceptional purity can be separated according to fluorescent markers using flow cytometry. High speed observations on large numbers of individual vesicles allows identification of subpopulations, and statistical comparison, within a single heterogeneous sample.

Animals↗

PubMed1997

Chromosomal and DNA ploidy characterization of salivary gland neoplasms by combined FISH and flow cytometry.

Concurrent DNA ploidy by flow cytometry and interphase FISH analysis of chromosomes 6 through 12, 17, 18, X, and Y were prospectively performed on 22 salivary gland neoplasms (four benign and 18 malignant) to investigate the diagnostic and biological implications of their alterations in these neoplasms. Our results show that benign neoplasms lack DNA aneuploidy and numerical chromosomal abnormalities. Low-grade malignant neoplasms, except for two lesions, manifested small chromosomal gains and losses and were generally DNA diploid or near-diploid aneuploid, whereas all high-grade tumors showed marked polysomy and were DNA aneuploid. Marked intratumoral and intertumoral chromosomal heterogeneity also were noted in and between individual tumors. Although polysomy was the main finding in DNA aneuploid lesions, monosomy was more noted in DNA diploid neoplasms and was restricted to chromosomes 8, 11, and 17. Significant correlation between the DNA index, chromosomal aneusomy, histological grade, and tumor stage was noted. Our study indicates that (1) benign salivary gland neoplasms lack gross DNA content and numerical chromosomal abnormalities, (2) clonal chromosomal alterations are manifested in most DNA diploid and all DNA aneuploid malignant tumors, (3) chromosomal gain is the most common alteration; chromosomal loss is less frequent and restricted to certain chromosomes, and (4) DNA aneuploidy and chromosomal aneusomy characterize tumors with aggressive features.

Adult↗

PubMed1999

Detection of minimal residual disease (MRD) after bone marrow transplantation (BMT) by multi-parameter flow cytometry (MPFC).

Multi-parameter flow cytometry (MPFC) was used to detect minimal residual disease (MRD) following bone marrow transplantation (BMT) in 21 patients. Bone marrow (BM)was analyzed pre-transplant and 3-4 months post-BMT while the patients were in clinical and morphological remission. MRD was detected by identifying cells with aberrant antigen expression and/or leukemia-associated phenotype (LAP) using MPFC. Prior to BMT, 8 out of 21 patients exhibited normal antigen expression based on normal BM samples while 13 BM aspirates had abnormal MPFC. Pre-BMT MPFC was abnormal in all 10 patients who were not in complete remission (CR) (>5% blasts in BM) as well as 3 patients acute lymphoblastic leukemia (ALL) who were in CR. In BM from ALL patients, an abnormal uniform B cell population was observed however antigen expression patterns varied greatly between patients. BM from acute myeloblastic leukemia (AML) patients showed an abnormal distribution of CD34+ cells. In addition, a correlation was observed between pre-BMT cytogenetics and MPFC. Only 2 out of 8 (25%) patients with normal MPFC pre-autologous bone marrow transplantation (ABMT) relapsed (AML), while 6 out of 13 (46%) patients with abnormal pre-BMT MPFC relapsed including 2 out of 3 patients who were transplanted in clinical CR. Pre-BMT MPFC may thus be an effective tool for detection of MRD by detection of a pre-transplant MPFC abnormality.

Adolescent↗