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Results for “Flow Cytometry”

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PubMed1998

Characterization of lymphocyte subpopulations in periapical lesions by flow cytometry.

The mechanisms by which the bacterial root-canal infection leads to periapical bone destruction (cysts or granulomas) are not yet well understood. Previous works have shown elements of an active immune response in the lesions. In the present study, flow cytometry was used to improve the characterization of immune cells. Semiquantitative immunohistochemical analysis showed the presence of plasma cells, macrophages and B and T cells. The simultaneous use of several antibodies in flow cytometry allowed a more precise phenotype of the lymphocytes. The cysts displayed an abundance of B lymphocytes at the same time as a relative scarcity of CD8+ cells. CD4+ lymphocytes were the dominant lymphocyte population in most cases. A small number of gamma delta T lymphocytes and natural killer cells was found. These preliminary results show that flow cytometry may be used to characterize immune cells from inflamed tissue and opens the possibility for further functional studies.

Adult↗

PubMed1990

Deoxyribonucleic acid ploidy and cell cycle analysis of colorectal carcinoma by flow cytometry. A prospective study of 137 cases using fresh whole cell suspensions.

Several retrospective studies suggest that abnormal deoxyribonucleic acid (DNA) content in colorectal carcinoma correlates with adverse clinical outcome. Many of these studies have used naked nuclei retrieved from formalin-fixed, paraffin-embedded tissues for flow cytometry. The purpose of this study was to prospectively analyze 137 colorectal carcinomas using fresh whole-cell suspensions for flow cytometry and to determine whether abnormal DNA content (DNA aneuploidy or tumors with high proliferative activity) correlates with Dukes' stage, histologic grade, lymphocytic infiltration of the tumor, tumor fibrosis, extramural venous spread, or tumor size. Cell suspensions for flow cytometry were prepared by enzyme disaggregation with collagenase XI, DNase, and trypsin. Satisfactory DNA histograms were obtained from 132 of the 137 samples. The mean coefficients of variance for the G1/G0 of the external 2C control, internal 2C populations, and aneuploid populations were 2.5, 3.5, and 3.5, respectively. The mean percentage of viable cells was 97%. Of 132 cases, 102 (77%) demonstrated abnormal DNA histograms, of which 77 (58%) showed DNA aneuploidy. Abnormal DNA histograms of DNA aneuploidy did not correlate with Dukes' stage. Tumors of higher histologic grade were more likely to demonstrate DNA aneuploidy, however, these differences did not reach statistical significance. The authors conclude that (1) satisfactory DNA histograms can be obtained with the use of a fresh, whole-cell technique; (2) abnormal DNA histograms did not statistically correlate with standard clinical, grading, or staging parameters; and (3) carcinomas of high histologic grade showed an increased proportion of aneuploid DNA histograms, but this trend did not reach statistical significance.

Adenocarcinoma↗

PubMed2006

Use of flow cytometry for analysis of phage-mediated killing of Enterobacter aerogenes.

In this study, the use of flow cytometry to analyze phage-mediated killing of Enterobacter aerogenes under varying conditions of temperature and nutrient availability was assessed. Bacteriophage UZ1, specific for an E. aerogenes strain, was applied at a multiplicity of infection (MOI) of 1 and 1000 to a Teflon surface, artificially infected with its host at a level of 4.5 log cells. After incubation for 20 h, bacteriophages were quantified using the soft agar layer method. For the quantification of bacterial cells, plate counting and flow cytometric analysis of live/dead stained cells were performed in parallel. At an MOI of 1, phage treatment was successful only after incubation under nutrient-rich conditions at 37 degrees C: E. aerogenes cells were not detected and a tenfold increase in phage UZ1 was observed. At a MOI of 1000, no E. aerogenes cells could be cultured after incubation at 37 and 4 degrees C. However, flow cytometric analysis revealed that lysis did not occur at 4 degrees C but was achieved during subsequent plate culture. In conclusion, the use of flow cytometry enabled identification of culture-based bias during plate culture. The flow cytometric assay used in this study proved to be rapid, as this culture-independent method does not require lengthy incubation periods post-sampling. The bacteriophage-mediated killing of E. aerogenes cells on Teflon surfaces indicated that disinfection of E. aerogenes with bacteriophage UZ1 can be successful when high MOIs are achieved, while at low multiplicities of infection conditions favorable for phage replication are required.

Bacteriolysis↗

PubMedDate not supplied

Application of flow cytometry in chronobiology.

A survey is given on flow cytometric techniques and their applications in chronobiology. Rapid automatic single cell measurements with a high rate (1,000-5,000 single cells/sec) enable the accumulation of large numbers of data at short intervals, thus obtaining important knowledge on circadian variations of cell proliferation in epidermis and other types of surface epithelium as well as hemopoiesis. In addition, the method has to some extent been used for monitoring cancer chemotherapy, and is thus available for the application in chronotherapy of malignancy. The introduction of rapid automatic techniques such as flow cytometry is a great advantage for any study on single cells and tissues where rhythmic variations in different functions have to be taken into consideration.

Animals↗

PubMed2000

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy.

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.

Anthraquinones↗

PubMed1980

The effect of the glutamine analog, AT-125, on the cell cycle of MCF-7 and BT-20 human breast carcinoma cells using DNA flow cytometry.

It was found by DNA flow cytometry that AT-125 preferentially inhibited the cell cycle progression in G1-phase of BT-20 more so than MCF-7 breast carcinoma cells in vitro and that cells washed free of the drug now possessed S-phase DNA content. It was also found that gamma-glutamyl transpeptidase levels were more than 2 times higher in BT-20 than in MCF-7 cells.

Antineoplastic Agents↗

PubMed2004

Frequency of proteinase 3 (PR3)-specific autoreactive T cells determined by cytokine flow cytometry in Wegener's granulomatosis.

OBJECTIVE: Previous studies have shown proliferation to Wegener's autoantigen, proteinase 3 (PR3). We tested immunogenicity of PR3-derived peptides and determined frequencies of PR3-specific T cells using cytokine flow cytometry in Wegener's granulomatois (WG). METHODS: Peripheral blood T-cell responses were measured after stimulation with previously described PR3-derived peptides. PBMC were stimulated with PR3-derived peptides or control stimuli for up to 10 days. Cells were stained with antibodies against CD4 or CD8, CD69 and intracellular TNF-alpha and analyzed by flow cytometry. PR3-specific T cells were counted as the percentage of CD69(+)TNF-alpha(+)double positive T cells after stimulation. RESULTS: Frequencies of PR3-specific peripheral blood T cells after short-term stimulation (<or=0.4%) were lower compared to frequencies of PR3-specific CD4(+)T cells (0.64+/-0.09%, mean+/-SEM) and PR3-specific CD8(+)T cells (0.65+/-0.18%) after 10 days of stimulation in WG. There were no differences of the frequency of PR3-specific T cells between WG and healthy controls after stimulation with other peptides. The frequency of PR3-specific CD8(+)T cells stimulated with a preferentially HLA-A*0201 binding PR3-peptide sequence was higher compared to the frequency of T cells stimulated with a HLA-B*0702 binding PR3-peptide in one WG patient whose HLA type was known (A2B7). CONCLUSION: Low frequencies of TNF-alpha(+)PR3-specific T cells can be detected in individual WG patients and controls using cytokine flow cytometry. The pattern and time course of cytokine production in response to PR3 peptides needs to be further elucidated. Additional factors such as the influence of proinflammatory or regulatory T cells might be important for the induction of autoimmunity in WG.

Amino Acid Sequence↗

PubMed2003

Comparison of comet assay, electron microscopy, and flow cytometry for detection of apoptosis.

Differentiating apoptosis from necrosis is a challenge in single cells and in parenchymal tissues. The techniques available, including in situ TUNEL (Terminal deoxyribonucleotide transferase-mediated dUTP-X Nick End-Labeling) staining, DNA ladder assay, and flow cytometry, suffer from low sensitivity or from a high false-positive rate. This study, using a Jurkat cell model, initially evaluated the specificity of the neutral comet assay and flow cytometry compared to the gold standard, electron microscopy, for detection of apoptosis and necrosis. Neutral comet assay distinguished apoptosis from necrosis in Jurkat cells, as evidenced by the increased comet score in apoptotic cells and the almost zero comet score in necrotic cells. These findings were consistent with those of electron microscopy and flow cytometry. Furthermore, using rats with burn or ischemia/reperfusion injury, well-established models of skeletal and cardiac muscle tissue apoptosis, respectively, we applied the comet assay to detect apoptosis in these muscles. Neutral comet assay was able to detect apoptotic changes in both models. In the muscle samples from rats with burn or ischemia-reperfusion injury, the comet score was higher than that of muscle samples from their respective controls. These studies confirm the consistency of the comet assay for detection of apoptosis in single cells and provide evidence for its applicability as an additional method to detect apoptosis in parenchymal cells.

Animals↗

PubMed2003

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry.

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time. STUDY DESIGN AND METHODS: Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model. RESULTS: The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one. CONCLUSION: A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.

Agglutination↗

PubMed1990

Gating of the so-called 'lymphocytic' cell population for the quantification of natural killer cells (CD16+) by flow cytometry causes loss of CD16 positive cells.

Natural killer cells can phenotypically be identified as CD16 positive with a specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.

Adult↗

PubMed1982

Application of flow cytometry to studies of pathogenic free-living amoebae.

Species of small, free-living amoebae of the genera Naegleria and Acanthamoeba can cause fatal amoebic meningoencephalitis. Previous investigations have shown that pathogenic amoebae are associated with thermally altered water. Flow cytometric techniques for identifying species of pathogenic and nonpathogenic amoebae from such water have been developed, using immunofluorescence and fluorescein-bound concanavalin A. Flow cytometry is accomplished with a cytofluorograph, in which cells are dispersed in a suspended carrier liquid and passed in front of a focused argon ion laser beam. Cells are then distinguished by the degree of scattered light (size) or fluorescence. Flow cytometry techniques have proven efficient for environmental samples, as indicated by the identification of pathogenic Naegleria fowleri and nonpathogenic Naegleri gruberi and Acanthamoeba castellanii isolated from the Savannah River Plant in South Carolina. Cytofluorographic analysis of environmental samples has several advantages over the current methods of isolation and classification of free-living amoebae. With this system, it is possible to rapidly identify species and quantitate mixtures of pathogenic amoebae in environmental samples. Cytofluorographic analysis of amoebic isolates reduces the time presently required to screen environmental sites for pathogenic amoebae. The cytofluorograph permits detection and species identification of nonthermophilic Naegleria spp. and Acanthamoeba spp. that could not easily be isolated for species identification by conventional methods. Other advantages of flow cytometry over fluorescent microscopy include a high degree of statistical precision due to the large numbers measured, high immunofluorescent titers, and elimination of subjectivity and fluorescence fading.

Agglutination Tests↗

PubMed1994

Evaluation of testicular germ cell tumor proliferation using DNA flow cytometry and nucleolar organizer silver staining.

We studied 41 testicular germ cell tumors (TGCT) using DNA flow cytometry and argyrophilic (silver staining) nucleolar organizer region (Ag-NOR) quantitation to evaluate the proliferative characteristics of TGCT and the significance of those analytical techniques. In nonseminomatous germ cell tumor (NSGCT), the percentage of S + G2M phase cells (%S + G2M) in DNA aneuploid tumor was higher than in DNA of diploid tumors. Likewise, in NSGCT the aneuploid Ag-NOR sites per nucleus (Ag-NOR count) were significantly higher in DNA aneuploid than in DNA diploid tumors. There was also a significant linear correlation between % S + G2M phase cells and the Ag-NOR count in TGCT. However, the DNA ploidy pattern and the % S + G2M phase cells, and Ag-NOR counts, did not correlate significantly with the patient survival rate or clinical stage. In conclusion, our study suggests that Ag-NOR enumeration is an effective technique for assessing cellular proliferative activity that is somewhat less expensive than DNA flow cytometry.

Cell Division↗

PubMed1996

Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry.

A novel culture system using biotin/streptavidin and flow cytometry was developed to compare maturation and growth rates in Plasmodium falciparum malaria parasites in two distinct red blood cell (RBC) populations. Biotin was used to label a selected RBC population which was then mixed with another distinct unbiotinylated RBC population. P. falciparum-infected RBCs were used to initiate co-cultures followed over 2-3 schizogonic growth cycles. Co-cultures were harvested and stained with streptavidin-fluorescein isothiocyanate (FITC) followed by fixation and staining of parasite DNA. The combination of biotin/streptavidin-FITC and DNA fluorochrome enabled simultaneous flow cytometric analysis of the two different RBC populations and of the parasitemias in each RBC population. We then used this system to study the in vitro susceptibility of RBCs from individuals with hemoglobin H (Hb H) disease to infection and growth of P. falciparum. Significant reduction in parasite multiplication was found in Hb H RBCs as compared with that in normal RBCs. This novel malaria culture system offers two major innovations: a method to compare directly the relative ability of any two red blood cell populations to support malaria parasite invasion and development under identical conditions, and a critical reduction in the volume of blood and reagents needed to assess parasite growth. The application of biotin-labeled RBCs in the flow cytometric analysis of parasite development may offer new insights in studies of the relationship between RBC defects and susceptibility to malaria parasites.

Animals↗

PubMedDate not supplied

Adherence of Helicobacter pylori to gastric carcinoma cells: analysis by flow cytometry.

An in vitro assay using immunofluorescence and flow cytometry for quantitative assessment of the adherence of Helicobacter pylori to cultured human gastric carcinoma (KATO III) cells was developed. Adherence was rapid, saturable, energy dependent, mannose resistant, and significantly inhibited by fetuin, a glycoprotein containing N-acetylneuraminyllactose. Pretreatment of KATO cells with neuraminidase from Clostridium perfringens, however, did not reduce adherence of H. pylori. Ultrastructurally, adherent cells of H. pylori were associated with indentations of KATO cell membranes. KATO cells should prove useful in the investigation of mechanisms of adherence of H. pylori to mammalian cells. Ultimately, this flow cytometric assay may be helpful in assessment of the adherence of laboratory strains of H. pylori directly to surface mucous cells dissociated from biopsied human gastric tissue.

Bacterial Adhesion↗

PubMed2004

[Immunophenotype analysis in 121 patients with lymphoproliferative diseases by flow cytometry].

BACKGROUND & OBJECTIVE: Multiple parameter immunophenotype analysis by flow cytometry (FCM) could improve the accuracy of diagnosis in lymphoproliferative diseases. This study was to analyze the immunophenotype of 135 samples, including lymph nodes, blood, bone marrow, and cerebrospinal fluid samples, from 121 patients with suspected lymphoid malignancies, to evaluate its role in diagnosis. METHODS: All samples were tested by routine morphological, pathological, and immunohistochemical methods, and analyzed by FCM in suspended single cells, to compare the accuracy of different diagnostic methods. RESULTS: (1) Three of 23 lymph nodes, which failed to be diagnosed by routine methods, were determined by flow cytometric immunophenotype analysis. (2) According to new WHO classification, 96 of 97 blood or bone marrow aspiration samples were diagnosed by flow cytometric immunophenotype analysis, while 88 of 97 samples were diagnosed by routine immunohistochemical method. (3) Multiple parameter immunophenotype analysis in cerebrospinal fluid samples by FCM improved the diagnostic accuracy of leukemia or lymphoma involvement of central nervous system. CONCLUSIONS: Multiple parameter immunophenotype analysis by FCM improves the accuracy of diagnosis in lymphoid malignancies, and can be used in diagnosis, differentiated diagnosis, and detection of minimal residue in lymphoproliferative diseases.

Adolescent↗

PubMed1991

The use of flow cytometry to detect the biosynthesis of complement components.

Agarose beads, an activator of complement, were incubated with MRC-5 or He 9 fibroblast cell lines under serum-free conditions. The beads were tested for binding of anti-complement antibodies by flow cytometry with a FACS 440 using FITC-labelled anti-Ig detection antibodies. Controls consisted of co-cultured beads incubated with irrelevant antibody or albumin, beads maintained in cell cultures containing cycloheximide, and beads which were not exposed to cells. The histograms demonstrated positive staining with anti-C3c, -C5, -C7 and -C9, but not with anti-C6 and -C8. Flow cytometry with multiple histogram analysis confirmed that the differences between the positive curves and the controls were statistically significant. The results show that cell-derived complement components (C3, C5, C7 and C9) were deposited on the beads and could be detected by flow cytometry.

Cell Line↗

PubMed1988

Barrett's esophagus. Correlation between mucin histochemistry, flow cytometry, and histologic diagnosis for predicting increased cancer risk.

A predominance of sulfated mucin in the nongoblet columnar cells of Barrett's specialized metaplastic epithelium has been postulated to be a form of mild dysplasia and to indicate an increased risk of adenocarcinoma. Flow cytometry for the analysis of nuclear DNA content and cell cycle parameters has also been postulated to be an objective aid in the diagnosis of dysplasia and carcinoma in Barrett's esophagus. The authors investigated the relationship among sulfated mucin, flow cytometric data, and histologic diagnosis in each of 152 biopsies from 42 patients who had Barrett's specialized metaplastic epithelium. Sulfated mucin, as detected by the high iron diamine-Alcian blue stain, was present in biopsies from 8 of 11 (73%) patients with the histologic diagnosis of dysplasia or carcinoma, in 7 of 9 (78%) patients whose biopsies were indefinite for dysplasia, and in 12 of 22 (55%) patients whose biopsies were negative for dysplasia (P = 0.37). Sulfated mucins predominated in 9%, 22%, and 9% of the patients, respectively (P = 0.56). Abnormal flow cytometry (aneuploidy or increased G2/tetraploid fraction) was found in all patients with the histologic diagnosis of dysplasia or carcinoma, in 3 of 9 (33%) indefinite for dysplasia, and in 1 of 22 (5%) negative for dysplasia (P = less than 0.0001). Neither the presence nor the predominance of sulfated mucin in the specialized metaplastic epithelium of Barrett's esophagus has sufficiently high sensitivity or specificity for dysplasia or carcinoma to be of value in managing patients. Abnormal flow cytometry shows excellent correlation with the histologic diagnosis of dysplasia and carcinoma; it detects a subset of patients whose biopsies are histologically indefinite or negative for dysplasia, but who have flow cytometric abnormalities similar to those otherwise seen only in dysplasia and carcinoma.

Barrett Esophagus↗

PubMed1985

Application of flow cytometry to detection and characterization of Legionella spp.

Flow cytometry, using fluorescein-bound specific antibodies and propidium iodide, was shown to be effective in detecting Legionella spp. in cooling tower waters. The procedure was quicker and less labor intensive than fluorescent microscopy. The use of these procedures also identified qualitative differences, perhaps related to infectivity, in Legionella populations.

Air Conditioning↗