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Results for “Flow Cytometry”

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PubMed2000

One-round determination of seven leukocyte subsets in rhesus macaque blood by flow cytometry.

BACKGROUND: Rhesus macaques are frequently used in biomedical research as experimental models for studying infectious diseases and for preclinical vaccination trials. The infection of these monkeys with simian immunodeficiency viruses (SIV) or simian-human immunodeficiency viruses (SHIV) reproduces the clinical and immunological characteristics of human infection by human immunodeficiency virus (HIV). Evolution of the immune response in the infected animals is generally analyzed by determining the lymphocyte subsets on blood samples using flow cytometry but requiring multiple, blood consuming, determinations. METHODS: Cell subsets present in whole-blood samples were labeled with a combination of anti-human monoclonal antibodies to CD2, CD20, CD4, CD8, and CD14 coupled to FITC or PE and analyzed by flow cytometry. RESULTS: In one round, we obtained the precise determination of macaque blood cell composition by flow cytometry. Monocytes, granulocytes, eosinophils, B lymphocytes, helper, and cytotoxic T lymphocytes were distinguished. Results obtained correlated strongly with those obtained with conventional blood cell differential systems and with separate staining of lymphocytes. The analysis of blood from healthy rhesus macaques and SHIV-infected animals demonstrated the accuracy of the determination even in very pathological situations such as macaques with simian AIDS. CONCLUSIONS: Our method allows fast determination of the blood cell composition and will be particularly useful to evaluate the cell subset evolution of macaques involved in large-scale experimental trials.

Animals↗

PubMed2003

Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry.

Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies. Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems. In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry. The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs's method, and the Yamane procedure. Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet. All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M. tuberculosis H37Ra. Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis. Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations. The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M. tuberculosis H37Ra. While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M. tuberculosis.

Analysis of Variance↗

PubMed1995

Rickettsia tsutsugamushi infection in cell culture: antibiotic susceptibility determined by flow cytometry.

Recent unpublished reports from northern Thailand of severe and sometimes fatal cases of scrub typhus, despite appropriate antibiotic therapy, suggest that resistance may occur. Current antibiotic susceptibility methods that use direct microscopic counts of Giemsa-stained cells or mouse protection assays are slow, labor-intensive, and expensive. We explored the use of flow cytometry to measure rickettsial infection in vitro in L-929 cells treated with and without doxycycline, ciprofloxacin, erythromycin, and chloramphenicol. It was possible to detect the rickettsiae down to a level of 83% of the cells infected, mean of 37 rickettsiae per cell, and 40% of cells with too many rickettsiae to count. This level of sensitivity was sufficient to determine the inhibitory effect of all four drugs at standard screening concentrations. At lower concentrations of doxycycline, flow cytometry detected inhibition of rickettsial growth at a concentration of 6.25 x 10(-2) micrograms/ml but not at 6.25 x 10(-3) micrograms/ml, suggesting that the minimum inhibitory concentration is somewhere between these two values. The data from this study show that flow cytometry permits the rapid screening of numerous rickettsial isolates for their susceptibility to a variety of antibiotics, but that visual counts of infected cells provide a more precise indication of rickettsial growth.

Animals↗

PubMed2006

Application of Gleason analogous grading system and flow cytometry DNA analysis in a novel knock-in mouse prostate cancer model.

OBJECTIVE: A new knock-in mouse adenocarcinoma prostate model (KIMAP) was established, which showed a close to human kinetics of tumour development. This study used a new mouse histological grading system similar to the human Gleason grading system and flow cytometry DNA analysis to measure and compare the new KIMAP model with human CaP and transgenic mouse adenocarcinoma prostate (TGMAP) model. METHODS: According to heterogeneity of the clinical standard for prostate cancer diagnosis, a close to human mouse standard for histological grading and scoring system, Gleason analogous grading system, was established in this study. Sixty KIMAP and 48 TGMAP prostate cancer samples were measured and compared with human CaP. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from both TGMAP and KIMAP models. RESULTS: Mice with CaP from KIMAP (n = 60) and TGMAP (n = 48) models showed a different distribution of histological scores (p = 0.000). KIMAP mice showed higher percentage (53.3%) of compound histological score rate than TGMAP (25%), but closer to the human clinical average (50%), which showed significant correlation with age (p = 0.001), while TGMAP mice showed unbalanced and random score distribution in all age groups. Flow cytometry analyses showed that most tumour tissues in KIMAP were diploid, analogous to the human condition, while all the TGMAP mice showed aneuploid tumours. CONCLUSIONS: Results of this study further show that KIMAP, a new generation of murine prostate cancer model, could be used as a supplementary model in addition to the currently widely used transgenic models.

Adenocarcinoma↗

PubMed2003

Comparison of the effect of mitochondrial inhibitors on mitochondrial membrane potential in two different cell lines using flow cytometry and spectrofluorometry.

BACKGROUND: Determination of mitochondrial membrane potential (DeltaPsim) is widely used to characterize cellular metabolism, viability, and apoptosis. Changes of DeltaPsim induced by inhibitors of oxidative phosphorylation characterize respective contributions of mitochondria and glycolysis to adenosine triphosphate (ATP) synthesis. METHODS: DeltaPsim in BSC-40 and HeLa G cell lines was determined by flow cytometry and spectrofluorometry. Its changes induced by specific mitochondrial inhibitors were evaluated using 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), tetramethylrhodamine ethyl ester, and MitoTracker Red. Mitochondrial function was further characterized by oxygen consumption. RESULTS: Inhibition of respiration by antimycin A or uncoupling of mitochondria by FCCP decreased DeltaPsim in both cell lines. Inhibition of ATP production by oligomycin or atractyloside induced a moderate decrease of DeltaPsim in HeLa G cells and an increase of DeltaPsim in BSC-40 cells. Statistically significant differences in DeltaPsim between the two cell lines were found with both flow cytometry and spectrofluorometry. Respirometry showed higher basal and FCCP-stimulated respiration in BSC-40 cells. CONCLUSION: Changes of DeltaPsim and oxygen consumption showed that BSC-40 cells are more sensitive than HeLa G cells to inhibitors of mitochondrial function, suggesting that BSC-40 cells are more dependent than HeLa G cells on aerobic ATP production. Determination of DeltaPsim changes by flow cytometry exhibited greater sensitivity than the ones by spectrofluorometry.

Animals↗

PubMedDate not supplied

Fluorescent probes and flow cytometry to assess rat sperm integrity and mitochondrial function.

Fluorescent assessment of cellular integrity and mitochondrial function by flow cytometry can provide a rapid and precise means of determining the functional status of large numbers of spermatozoa. In the present study, rat sperm viability was assessed with SYBR-14 and PI and sperm mitochondria were differentially labeled with JC-1. Sperm samples of variable viability were prepared using varying proportions of fresh and frozen spermatozoa. SYBR-14 stained sperm correlated well with expected sperm viability (r = 0.98). Motile sperm stained with JC-1 appeared orange in the midpiece indicating a high mitochondrial membrane potential whereas immotile sperm with a low membrane potential stained green. The percentage of spermatozoa staining orange was highly correlated (r = 0.99) with expected sperm viability. Flow cytometry using specific fluorescent probes is a useful technique for detecting changes in rat sperm plasma membrane integrity and mitochondrial function in large numbers of spermatozoa.

Animals↗

PubMed1994

Double fluorescence analysis of human cytomegalovirus (HCMV) infected human fibroblast cultures by flow cytometry: increase of class I MHC expression on uninfected cells and decrease on infected cells.

Cultured human foreskin fibroblasts (HFF) were infected with different multiplicities of infection (moi 0.001-0.1) of human cytomegalovirus (HCMV) strain AD 169 or a clinical isolate. Percentage of infected cells was determined by analysis of immediate early (IEA), early (EA), and late (LA) virus antigen expression with flow cytometry or by immunoperoxidase staining. Changes in the expression of class I MHC surface molecules were demonstrated by comparing the mean fluorescence intensities of infected HFF cultures with those of mock infected cell cultures by flow cytometry. At day three post infection single fluorescence analysis showed that infected HFF cultures split into low and high density class I MHC bearing cells. The addition of anti-interferon beta reduced the expression of class I MHC, distinctly. The assumption that infected cells down-regulate and uninfected cells up-regulate their expression of class I MHC molecules was demonstrated by double fluorescence analysis both with flow cytometry and fluorescence microscopy. Analysis of class I MHC-antigen expression versus immediate (IEA, mab E13), early (EA, mab 9221), or late (LA, mab BM219) virus antigen expression yielded three cell populations of HCMV infected HFF cultures three days post infection: 1. uninfected cells with an increase of class I MHC, 2. high density class I MHC, IEA and/or EA expressing cells, and 3. low class I MHC, IEA, EA and LA expressing cells.

Antibodies, Monoclonal↗

PubMed1988

Monoclonal antibodies to proliferating cell nuclear antigen (PCNA)/cyclin as probes for proliferating cells by immunofluorescence microscopy and flow cytometry.

Proliferating cell nuclear antigen (PCNA)/cyclin is an intranuclear polypeptide antigen that is found in both normal and transformed proliferating cells. We have recently described two mouse monoclonal antibodies reacting with PCNA. In this report we describe the application of these antibodies to the study of proliferating human cells by indirect immunofluorescence microscopy and by flow cytometry. A fixation/permeation procedure was developed in order to obtain satisfactory binding of monoclonal PCNA-specific antibodies to proliferating cells. This method involved fixation with 1% paraformaldehyde followed by methanol treatment. For the staining of cells in suspension with the IgM type monoclonal antibodies lysolecithin was added to the paraformaldehyde solution to achieve a better permeation by the antibody molecules. This procedure gave a good ratio of specific staining relative to the background staining. It also preserved the shape and normal architecture of the cells as judged by visual microscopic observation and by light scatter measurements using a flow cytometer. Furthermore, this fixation technique permits simultaneous labeling of DNA by propidium iodide and PCNA by monoclonal antibodies. PCNA was detected in various types of normal and transformed proliferating cells by indirect immunofluorescence. Quiescent peripheral blood mononuclear cells were PCNA-negative whereas a fraction of lectin-stimulated lymphocytes became PCNA-positive. Similarly, early passages of fetal skin fibroblasts were PCNA-positive but non-proliferating senescent fibroblasts of later passages were PCNA-negative. The association of PCNA-staining by monoclonal antibodies with cell proliferation was confirmed by flow cytometry. Simultaneous labeling of PCNA and DNA showed that the PCNA signal increased during the G1 phase of the cell cycle, reached its maximum in the S-phase, and declined during the G2/M phase. Using cell sorting we demonstrated that mitotic cells had a very low PCNA signal. Thus, monoclonal PCNA-specific antibodies offer a convenient tool for the detection of human cell proliferation by immunofluorescence microscopy and by flow cytometry.

Animals↗

PubMed2000

The preparation of periapical lesions for flow cytometry.

AIM: To devise an optimal protocol and to analyse the leucocyte composition of periapical (PA) lesions by flow cytometry. METHODOLOGY: PA lesions were mechanically agitated, with and without proteolysis. This was with either 0.2% collagenase alone, or in combination with 0.02% DNA-ase in serial incubations until all tissue was digested. The efficacy of each method was assessed by counting total cell yield and cell viability. Phenotype stability was gauged by the percentage of peripheral blood leucocytes (PBL) which expressed CD45RB, CD3, CD20, CD4 and CD8 before and after mechanical and collagenase treatment. RESULTS: Disaggregation of PA lesions was superior if collagenase was present, but cell clumping was problematic unless the DNA-ase was also added, and serial digestion with this combination produced optimal cell yield and viability. Nevertheless, the total number of cells released rarely exceeded 105, though viability was in excess of 80%. Mechanical agitation and proteolysis adversely affected PBL phenotypes, but collagenase digestion limited to 10 min caused least damage. Flow cytometric analysis of disaggregated PA lesions failed to identify more than 7.9% (mean, range 6-10%) CD45RB + cells. CONCLUSIONS: Because of the necessity for single cell suspensions, flow cytometry is not easily applied to the analysis of leucocytes in PA lesions, and further refinements in tissue disaggregation and cell preparation are required.

Antigens, CD↗

PubMedDate not supplied

Platelet function testing by flow cytometry.

The availability of fluorescent monoclonal antibodies and probes now provides a powerful tool for examining platelet function by flow cytometry. These techniques cna be employed to determine the resting and stimulated expression of platelet glycoprotein receptors, the activation status of platelets assessed by secretion of granule contents (including expression of activation-dependent neoantigens), adhesion ligand binding to platelets, and intracellular calcium flux after exposure of platelets to agonist. In addition, flow cytometry has now been used to study the functional properties of stimulated platelets, including microparticle generation, platelet aggregation, and platelet-heterotypic cell conjugate formation. This brief review is presented as a general outline of the literature that uses flow cytometric methodology to examine in vivo and ex vivo platelet function.

Blood Platelet Disorders↗

PubMed1999

Evaluation of the monocyte counting by the ABX Vega. Comparison with the manual method and fluoro-flow cytometry.

This study allowed evaluation and comparison of the precision (20 samples) and agreement (200 samples) of the relative and absolute values of the monocyte count using three methods: microscopic, flow cytometry and the automated ABX Vega hematology analyzer. Flow cytometry is the most precise method, even if the coefficient of variation of the automated analyzer is very similar. The coefficient of correlation between the ABX Vega and the flow cytometer is very satisfactory (r=0.8042). This study of the agreement also made it possible to confirm the difficulty that automated hematology analyzers have in differentiating between some granulocytes and monocytes and their propensity to overstimate the latter when the sample includes immature forms of granulocytes. This fact stresses the importance of the microscopic method, despite its lack of precision. The observed discrepancies did not lead to any difficulties in clinical interpretation; however, this finding should be taken into consideration, particularly in myleoproliferative syndromes and the laboratory monitoring of chemotherapy.

Flow Cytometry↗

PubMed1989

Characterization of liposome suspensions by flow cytometry.

Novel approaches to drug delivery and induction of immune responses using liposomes have received much attention in recent years. Liposomes, however, are not a singular entity, but can be produced with a diverse group of phospholipids that form microspheres of different sizes, physical structure, electrochemical characteristics, and most importantly, physiologic properties. The purpose of this study was to establish the usefulness of flow cytometry as a convenient, rapid method for assessing the relative size and uniformity of liposomal preparations. Liposomes were made from phospholipid suspensions by sonication alone, or sonication followed by microemulsification. Forward laser light scatter (FSC) analysis of liposomal preparations by flow cytometry indicated that microemulsification produced homogeneous, small vesicles which were less than 1 micron in diameter, compared to the more heterogeneous sized liposomes generated by sonication alone. Transmission electron micrographs of the liposomal preparations were used to confirm the FSC results and showed that liposomes prepared by microemulsification were homogeneous, unilamellar vesicles which exhibited a mean diameter of 99.8 nm, whereas the sonicated-only preparation was more heterogeneous in size, exhibiting a mean diameter of 154.1 nm. Analysis of various liposome preparations by FSC during a 9 week storage period showed that small vesicles were relatively stable. We conclude that flow cytometry using FSC analysis provides a rapid, reproducible and convenient method to evaluate the relative size, uniformity and stability of liposomes.

Animals↗

PubMed1988

Cell populations in human early pregnancy decidua: characterization and isolation of large granular lymphocytes by flow cytometry.

Cell populations of human pregnancy decidua, obtained by enzymic digestion from first trimester samples, were analysed by flow cytometry after labelling with monoclonal antibodies. The majority of these decidual cells (75%) were of bone marrow origin. The most abundant cell type expressed antigens characteristic of large granular lymphocytes (LGL), although macrophages and small numbers of classical T cells were also present. Three subsets of decidual LGL can be defined by single-and double-antibody labelling. Most decidual LGL are positive for NKH1, a marker of peripheral blood LGL, but negative for CD16, the Fc receptor of NK cells, and for the T-cell markers CD3 and CD5. About half the NKH1-positive cells also express CD2, associated with the E-rosette receptor, and are identical to the CD3-negative/CD2-positive cells reported previously in early pregnancy decidua. The NKH1-positive cells apparently correspond to a minor subset of peripheral blood LGL. The remaining decidual LGL are positive for CD16 and negative or only dimly positive for NKH1, and are similar to the major type of peripheral blood LGL. After purification by flow cytometry, the NKH1-positive cells were demonstrated to be of similar size to, but slightly higher granularity than, lymphocytes, whereas the CD16-positive cells were larger and more granular. The possible role of decidual LGL in modulating placental development is discussed.

Antibodies, Monoclonal↗

PubMed1995

Radial scar versus tubular carcinoma of the breast. A comparative study with quantitative techniques (morphometry, image- and flow cytometry).

The present study is focused on the differential diagnosis between radial scar (RS) and tubular carcinoma (TC) using morphometrical and cytophotometrical analysis (static and flow cytometry) of a number of histologically well-established RS cases (17 lesions) compared with 6 early infiltrating small TC with sclerotic stroma and pseudo-RS fields. One case displayed both RS and TC foci in contiguity. Mean nuclear area was larger in the group of tubular carcinomas (51.0 mu 2) than in the case of radial sclerosis (38.30 mu 2). We also found a larger number of aneuploid cases in tubular carcinomas measured by image cytometry, but both types of lesions were diploid when measured by flow cytometry; only one case of radial scar resulted aneuploid.

Adenocarcinoma↗

PubMed1989

Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: characterization of an assay in breast cancer cell lines.

Progesterone receptors (PR) are the strongest predictors of response to hormone therapy in metastatic breast cancer, while PR and the DNA indices of cell ploidy and percentage of S phase are useful prognostic indicators in early stage breast cancer. We have developed a flow cytometry method to measure PR and DNA indices simultaneously using two aneuploid breast cancer cell lines--PR-positive T47D cells and PR-negative MDA-231 cells. Cells were pretreated with the progestin 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione, harvested, counted, fixed with paraformaldehyde, and permeabilized with Triton X-100. To measure total PR, cells were first exposed to a mixture of the mouse anti-PR monoclonal antibody AB-52, which binds both Protein A and Protein B of human PR, and to monoclonal antibody B-30 or B-64, which bind only Protein B. Then the cells were treated with fluorescein isothiocyanate-conjugated goat anti-mouse second antibody to produce a green fluorescence signal corresponding to PR. To measure nonspecific binding, cells were treated with mouse IgG1 as the first antibody in a parallel incubation. Specific immunoassayable PR is the difference between total and nonspecific binding. Following the antibodies, the cells were treated with RNase A and propidium iodide to give a red fluorescence signal corresponding to DNA content. Red and green fluorescence per cell was then quantified by flow cytometry. This method gives a strong specific signal for PR in several T47D cell sublines but no specific binding in MDA-231 cells. Progestin treatment led to apparent increases in PR. The proportion of cells in the G0-G1, S, and G2-M phases of the cell cycle was determined from DNA histograms and showed that both cell lines were hyperdiploid. The simultaneous flow cytometry method allowed assignment of relative PR levels in subsets of cells segregated by their DNA content. In T47D cells, PR were present throughout the cell cycle, and levels doubled in G2 and mitosis.

Antibodies, Monoclonal↗

PubMed2004

Small lasers in flow cytometry.

Laser technology has made tremendous advances in recent years, particularly in the area of diode and diode-pumped solid state sources. Flow cytometry has been a direct beneficiary of these advances, as these small, low-maintenance, inexpensive lasers with reasonable power outputs are integrated into flow cytometers. In this chapter we review the contribution and potential of solid-state lasers to flow cytometry, and show several examples of these novel sources integrated into production flow cytometers. Technical details and critical parameters for successful application of these lasers for biomedical analysis are reviewed.

Flow Cytometry↗

PubMed1988

Hematoporphyrin/DAPI staining: simplified simultaneous one-step staining of DNA and cell protein and trial application in automated cytological screening by flow cytometry.

We describe a procedure for simplified, simultaneous one-step staining in 10 min for DNA and cell and tissue proteins using a newly developed staining solution containing 0.03% hematoporphyrin (HP) with 0.001% DAPI [or with Hoeschst 33342 (HO)]. These HP/DAPI or HP/HO solutions were especially developed to facilitate a trial of automated cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of DNA and that of protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of DNA and protein. On flow cytometry, a single UV beam (370 nm) from the argon laser was used for excitation of both dyes. Measurement of DNA was done using a 470-nm bandpass filter and of protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of protein and DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and cancer cells easier.

Animals↗

PubMed1989

DNA flow cytometry, clinical and morphological parameters as prognostic factors for advanced malignant and borderline ovarian tumors.

Patients with malignant ovarian (n = 111) and borderline (n = 8) tumors (FIGO stage III/IV) underwent surgery and chemotherapy and were analyzed clinically (age, residual tumor after surgery) and morphologically (type, grade, psammoma body content), and by means of flow cytometry (DNA ploidy, S-phase fraction). Follow-up was 12-72 months for investigation of survival. Patients under 60 years of age (n = 18) with malignant tumors showed longer survival than patients over 60 (n = 93) (P = 0.078). Residual tumor was relevant for prognosis in malignant tumors only if macroscopically there was no residual disease (n = 13). There were no significant differences between residual tumors less than or equal to 2 cm (n = 61) and greater than 2 cm (n = 37). WHO typing was of little importance for survival analysis. Compared to borderline tumors (n = 8), serous (n = 65), endometrioid (n = 13), nonclassifiable (n = 12), mucinous carcinomas (n = 8), and nonepithelial tumors (n = 12) had a poor prognosis. Psammoma bodies were found in 25 patients with serous carcinomas, 7 of them had a high content. The prognosis for these 7 patients was much better than that for patients with a moderate or low psammoma body content (P = 0.006). Twenty-three epithelial tumors were graded G1, 28 were G2, and 47 were G3. However, grading was considered only as a prognostic factor in serous carcinomas (n = 65) (P = 0.028). A total of 199 DNA histograms from 119 patients were analyzed by flow cytometry (FCM). There were no correlations between tumor type and DNA ploidy or S-phase fraction. Seven of eight borderline tumor and all serous carcinomas with a high content of psammoma bodies were diploid combined with a low (less than or equal to 4%) S-phase fraction. DNA ploidy and S-phase fraction were excellent prognosticators. Of 99 epithelial malignant tumors, 35 were diploid and 64 were aneuploid. An S-phase fraction less than or equal to 4% was found in 39 patients, 4.1-10% in 73 patients, and greater than 10% in 23 patients. Diploid tumors and tumors with a low S-phase fraction showed the best survival (P = 0.007, resp. 0.0001). Our study emphasizes the importance of an accurate histology, including information on psammoma body content, and the importance of DNA flow cytometry. The advantage of FCM is that the results are simple, reproducible, and objective.

Age Factors↗