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Results for “Flow Cytometry”

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PubMed1995

Sex determination in dioecious plants Melandrium album and M. rubrum using high-resolution flow cytometry.

High-resolution flow cytometry was used to analyze nuclear DNA content in dioecious plants Melandrium album and M. rubrum. Very low coefficients of variation (0.53-0.70%) of G0/1 peaks were obtained after the analysis of nuclei isolated by chopping leaf tissues. In both species, females have lower DNA content than males due to sex chromosome heteromorphism. This made it possible to discriminate peaks belonging to female and male nuclei on the same histogram and permitted unambiguous sex identification in both species. The results indicated the possibility of diagnosing sex also in other heterogametic dioecious plants. The assay described here might be interesting in any area where rapid sex identification is required at an early stage of plant development.

DNA, Plant↗

PubMed2003

Mapping T cell epitopes by flow cytometry.

Epitope mapping by flow cytometry is a very modern approach that not only identifies T-cell epitopes but simultaneously allows for detailed analysis of the responding T-cell subsets including lineage, activation marker expression, and other markers of interest. The most frequently used approach is based on the identification of intracellular cytokines in secretion-inhibited activated T cells following stimulation with peptides or peptide pools. A more recently developed assay analyzes T-cell proliferation by measuring the decrease in carboxyfluorescein diacetate succinimidyl ester staining in proliferated cells. This article includes information on peptide configuration, a section on the design and efficient application of peptide pools, and working laboratory protocols for both assays.

Epitope Mapping↗

PubMed1988

Modified histogram subtraction technique for analysis of flow cytometry data.

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time-consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel-by-channel histogram subtraction. Its accuracy is compared to that of the channel-by-channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram. The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.

Cell Separation↗

PubMed1986

Selective loss of suppressor T cells in rheumatoid arthritis patients: analysis of peripheral blood lymphocytes by 2-dimensional flow cytometry.

Two-colour flow cytometry analysis of the peripheral blood T cells in 28 patients with rheumatoid arthritis (RA) and healthy individuals showed a significant decrease in the percentage of the suppressor T cells (Leu 2a + Leu 15+) in severely active disease (5.9 +/- 2.1%) as compared to controls (13.4 +/- 3.8%) and active disease (12.9 +/- 5.9%). A significant adverse correlation was also observed between disease activity (Lansbury's index) and the percentage of suppressor T cells. Activated T cells (Leu 4+ HLA-DR+) were detected in a higher percentage and prevalence in the active group. Two-color immunofluorescence of T cell subsets may provide more accurate information concerning the immune imbalance of RA patients than the conventional single-color analysis.

Adult↗

PubMed1995

Competence effect of PDGF on Ki-67 antigen and DNA contents, and its inhibition by trichostatin-A and a butylydene phthalide BP-421 in primary smooth muscle cells of rat aorta by flow cytometry.

We investigated with flow cytometry the platelet-derived growth factor (PDGF)-induced competence in primary cultured smooth muscle cells (SMC) of rat thoracic aorta. A cytogram was obtained 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. (1) The nearly confluent SMC after 6 d-culture with 5% fetal bovine serum (FBS) were further cultured under serum starvation for 2d. (2) SMC was cultured with 5% FBS for 4 d, and then with 5% FBS+trichostatin-A (TS-A) (1 microgram/ml) for 2d. The cytogram showed the broadening of Ki-67 antigen signal for G0/G1 phase by TS-A compared with that of SMC under serum starvation, suggesting the existence of early and late phases of G1. (3) After serum starvation, the preculture with PDGF (100 ng/ml) for 3 h which was followed by a further 15 h-culture with 3% FBS caused significant more entry into S phase than control culture. The extent was greater than that with 15 h-culture with 10% FBS. (4) 15 h-culture with 1% FBS after PDGF (30 ng/ml) pretreatment stimulated entry into S phase cells, which was inhibited by TS-A (1 microgram/ml) and by a butylydene phthalide derivative BP-421 (3 micrograms/ml). Flow cytometric analysis demonstrate that PDGF pretreatment stimulates the entry into S phase by 20% of total SMC having early and late phases of G1, and that the PDGF-competence is inhibited by TS-A and BP-421.

Animals↗

PubMed1980

Bacterial growth studied by flow cytometry.

The feasibility of flow cytometry for measurements on bacteria has been demonstrated by measurements of DNA-associated fluorescence of Escherichia coli K-12 in various phases of cell growth. Bacteria were stained with a combination of ethidium bromide and mithramycin after fixation in 70% ethanol. Cultures grown to stationary phase accumulated in two peaks representing cells with two and four chromosomes. Qualitatively similar histograms were obtained with cells grown in the presence of chloramphenicol, whereas cells of the temperature sensitive strain E 177 (dnaA) ended up with only one chromosome per cell at the restrictive temperature. The fluorescence intensity of cells with one chromosome was about 10(3) times smaller than that of human diploid cells. Instrumental resolution at this level of intensity was CV = 5%, whereas peak widths corresponded to CV = 7-8%. Dyes bound to RNA did not appear to contribute significantly to the fluorescence.

Bacteriological Techniques↗

PubMed1999

Detection of intracellular phosphorylated STAT-1 by flow cytometry.

We have applied flow cytometry to the investigation of interferon-gamma activation of human monocytes. This approach uses monoclonal antibodies that distinguish between the native and phosphorylated forms of STAT-1. It enables rapid and quantitative assessment of STAT-1 phosphorylation on a discrete cell basis and is both more sensitive and less time consuming than immunoblotting. Furthermore, it allows for discrimination between a mixture of cells that differ in their response to interferon-gamma. This approach should allow for the evaluation of different intracellular signaling pathways using a combination of monoclonal reagents that are specific for native and activation modified proteins. Application of this form of testing should prove valuable in screening for signaling defects in selected patients with recurrent infections. In addition, this technique should permit dissection of a full range of cellular signaling pathways at the protein level.

DNA-Binding Proteins↗

PubMed1995

Usefulness of the scraping method for DNA flow cytometry in breast tumors.

Flow cytometric DNA analysis is an important prognostic tool in breast cancer. We evaluated the possibility of performing DNA analysis on cell suspensions obtained by scraping the cut surface of breast tumors; 31 breast tumor nodules, including six benign and 25 malignant lesions, were studied. From each case, cell suspensions acquired by mechanical mincing of a fresh frozen tissue fragment and by two different scrapings (central and peripheral) from the cut surface of the tumor were analyzed via flow cytometry. In all cases, comparison of the DNA histograms for three samples showed no significant differences in the appearance of debris or in the value of coefficient of variation of the G0-G1 peak. All benign nodules showed a normal DNA stemline in all specimens. In 23 of 25 cases of breast carcinoma, the ploidy of the three preparations was similar, with a concordance in 12/14 (85, 71%) cases in DNA nondiploid tumors. Linear regression analysis showed a good correlation in DNA index between either scraping sample and the tissue fragment (r = .955 and r = .905). The results indicate that the scraping technique provides excellent cell suspensions and DNA histograms comparable to those obtained from mechanical mincing of tissue fragments. The technique minimizes preparation time and avoids consuming much tissue and, thus, is the method of choice when very small cancers have to be analyzed.

Biopsy↗

PubMed1985

A simple and practical technic for detecting cancer cells in urine and urinary bladder washings by flow cytometry.

DNA measurement by flow cytometry has been demonstrated to be a potentially useful technic in the diagnosis of bladder cancer by detecting neoplastic cells in bladder washings and urine specimens. The authors' goal was to develop a simple and practical method utilizing the new generation of cytofluorographs designed for use in the clinical laboratory. This method combined direct fixation with cell lysis yielding fixed intact nuclei. Following RNase and pepsin digestion, the nuclei were separated from debris and aggregates on a sucrose barrier, stained with ethidium bromide, and analyzed with an argon laser analytic cytofluorograph. Urines and bladder washings from 14 patients with positive urinary cytology and histologically diagnosed bladder cancers were compared with specimens from patients without urothelial malignancies. DNA histograms clearly delineated aneuploid from diploid populations and often identified S, G2M, and G1 phase nuclei. Aneuploid populations have been detected in all tumor specimens with positive cytologies studied to date.

Aged↗

PubMed1991

Dicentric chromosome frequency analysis using slit-scan flow cytometry.

Slit-scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following gamma irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV gamma-rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit-scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear-quadratic manner according to the relation f(D) = 4.52 x 10(-5) + 5.72 x 10(-5) D + 1.19 x 10(-4) D2.

Algorithms↗

PubMed1994

Identification of CD34+ cord blood cells and their subpopulations in preterm and term neonates using three-color flow cytometry.

Three-color flow cytometry was used to identify CD34+ cord blood (CB) hematopoietic progenitors of preterm (n = 13) and term (n = 18) neonates. The frequency of CD34+ CB cells gated on the lymphocyte fraction in term neonates is about half the frequency of preterm neonates (mean 1.84 +/- 0.8 vs. 3.49 +/- 1.8, p < 0.005). The difference of absolute CD34+ cells between the 2 groups did not reach statistical significance (premature 169 +/- 161 vs. mature 108 +/- 62.6 x 10(6) cells/l). The proportion of myeloid lineage-committed CD34+ cells coexpressing the CD33 antigen in preterm neonates does not significantly differ from that found in term neonates. However, in preterm neonates a higher fraction of erythroid lineage-committed CD34+ cells coexpresses the CD71 antigen as compared with term neonates (mean 28.7 +/- 14.1 vs. 11.4 +/- 5.7, p < 0.001). The positive correlation between gestational age and the ratio of myeloid/erythroid lineage-specific progenitor cells (r = 0.61, n = 31, p < 0.0005) suggests gestational changes in lineage commitment of CD34+ cells.

Antigens, CD↗

PubMed1993

DNA analysis of head and neck squamous cell carcinoma by flow cytometry.

The use of flow cytometry to measure the DNA content from tumors has evolved over the years. In squamous cell carcinoma arising in the head and neck, there has not been uniform agreement in the literature, and decisions regarding patient treatment cannot be made using this parameter. The use of proliferating cell nuclear antigen (PCNA), a newly available marker of a cell's proliferative activity (S-phase fraction) is also discussed. In a prospective series of patients, the findings of diploidy, aneuploidy, low (PCNA) positivity, and high PCNA positivity are compared to known biological parameters. Strong trends are shown demonstrating biological aggressiveness associated with aneuploidy, high PCNA fraction, and the combination of aneuploidy and high PCNA fraction. The potential use of whole-cell preparation to determine ploidy and PCNA fraction as a predictor of metastatic potential are discussed. The whole-cell preparation technique allows accurate DNA ploidy measurements and, with the use of PCNA, a measure of proliferative activity. These parameters combined with known TNM staging may 1. allow alteration in treatment and ultimately affect patient survival, and 2. allow comparison of treatment modalities between biologically similar tumors.

Aged↗

PubMed1998

Flow cytometry for assessing biocompatibility.

Flow cytometry (FCM) was examined as a possible procedure for measuring in vitro the biocompatibility of implant materials for orthopedic and dental surgery. The human osteoblast-like cell line MG63 was grown on hydroxyapatite (HA) and P2O5 glass-reinforced HA composite discs and compared with the same cells grown on polystyrene culture dishes. While morphological observation at the light and electron microscopic levels showed no major deleterious effects, FCM indicated that cell size was somewhat reduced, particularly by growth on the HA composite. Morever, this material also appeared to delay the progression of the cells from the G0/G1 into the S phase of the cell cycle. In addition to this low level of inhibition of cell growth relative to control cultures, FCM analysis also demonstrated that the glass-reinforced HA caused some down-regulation of the expression of osteocalcin and fibronectin, two antigens which play a vital part in the integrity and function of bone and soft connective tissue, respectively. These results thus show, first, that although HA and the HA composite used in these experiments were generally biocompatible, they nevertheless had certain suboptimal effects on the cells; and second, that FCM could be a highly useful procedure for effectively screening and evaluating important biological responses to implant materials.

Biocompatible Materials↗

PubMed1994

Phenotypic differences among morphologically similar small-cell carcinomas detected with a panel of monoclonal antibodies and indirect immunofluorescence and flow cytometry.

Indirect immunofluorescence and flow cytometry were used to determine reactivity of a panel of 75 monoclonal antibodies (MAbs) and controls (provided by the Third International IASLC Workshop on Lung Tumor and Differentiation Antigens) with 3 morphologically similar prototype continuous-culture small-cell-carcinoma cell lines (SCC) (NCI-H69, NCI-H146, and NCI-H510). All cell lines had some reactivity with some of the MAbs. There is, however, differential expression of antigens amongst the prototype cell lines, which may provide a useful method for phenotyping and sub-classifying SCC. The reactivity of the 3 cell lines was greatest with MAbs in Clusters I, 1c, 2, 4, 6, and 9, and least with MAbs in clusters W7, 8, 13, 14, and W15, with few exceptions. Although morphologically similar, each of the SCC cell lines has a unique pattern of reactivity with the workshop MAbs. For example, although a control MAb, CD56 (NKHI), which identifies an epitope on NCAM (neural cell adhesion molecule) common among many SCC lines, stained more than 90% of cells in each of the prototype cell lines, one MAb of the current panel, SEN7, which also identifies a CD56 epitope on 15 SCC lines did not react as strongly with H-146 and H-510 as with H-69. If appropriately reactive MAbs can be identified for individual patients' tumors, they can be coupled to suitable radioisotopes or toxins for individualized patient treatment.

Antibodies, Monoclonal↗

PubMed1982

Characterization of bladder papilloma by two-parameter DNA-RNA flow cytometry.

Two-parameter flow cytometry (FCM) studies of 0.9% NaCl solution bladder irrigation specimens were performed on 48 patients with histologically orderly or atypical papilloma of the urinary bladder in order to assess the value of RNA as a possible second parameter, along with DNA, in the detection of bladder tumors. DNA, RNA, and nuclear diameter measurements were obtained for each of 5000 cells/sample, and analyses were based on the distributions of those values. With the use of DNA content alone, 22 cases (46%) were classified positive by FCM. With RNA content as an additional parameter, 40 cases (83%) were positive. Two cases were suspicious, and 6 cases were normal by both parameters. Of 28 patients with papillomas showing histological atypia, 16 patients had positive DNA histograms, including 3 patients with aneuploid stemlines, but 24 of the 28 patients had positive RNA histograms. Of 20 patients with orderly papillomas, 6 patients had positive DNA histograms, including 3 patients with aneuploid DNA stem cell lines, but 16 of the 20 patients had positive RNA histograms. Thus, the probability of positive DNA histograms is higher in atypical papillomas (57%) than in orderly papillomas (30%), whereas elevated (positive) RNA is more characteristic of all papillomas without distinction between those that are histologically atypical (86% positive) or orderly (80% positive). For patients at risk of developing papillary bladder tumors, two-parameter DNA-RNA FCM appears to offer greater diagnostic sensitivity than does FCM based on DNA content alone.

Cell Nucleus↗

PubMed1999

Adaptive responses of Ralstonia eutropha to feast and famine conditions analysed by flow cytometry.

Results obtained by flow cytometry allow conclusions to be drawn about how the physiological states of Ralstsonia eutropha JMP134 are connected with survival strategies under distinct growth conditions. During both feast and famine conditions the cells were found to proceed through sharply separated phases of life. Two sources of carbon and energy, one poor (0.02% phenol) and one rich (0.2% pyruvate and 0.1% yeast extract) were chosen to study the cellular responses. Despite the major differences in carbon source, when growth stages of the bacteria on the two substrates were characterised in batch growth, only minor differences were found in the time course of the membrane potential related fluorescence intensity (MPRFI). This also applied to the rRNA content and the size-correlated forward scatter (FSC) signal of the cells, both of which increased to high levels during the (early) exponential growth phase. On the rich medium, DNA synthesis initially occurred in an uncoupled manner, then a high rate of PHB formation followed when nutrients began to be limiting. Under famine conditions, the cellular responses were much more complex. PHB was synthesised, then DNA synthesis occurred in a 'eukaryotic' mode, to be succeeded by renewed PHB synthesis. To obtain defined cell physiological states, the chemostat technique was used in addition to batch experiments. The results obtained clearly indicated that key events in cell physiology, including initiation of DNA replication and overflow metabolism, occurred in a hierarchically ordered manner and were tightly correlated with changes in the environmental conditions of the bacterial cells.

Adaptation, Physiological↗

PubMed2002

Quantitation of baculovirus particles by flow cytometry.

A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.

Animals↗

PubMed1991

Enrichment of subpopulations of respiratory epithelial cells using flow cytometry.

The application of flow cytometry to enrich airway epithelial cell subpopulations is described. A complementary epithelial cell preparative technique is also outlined. The ability of the airway epithelium to protect the lung from environmental insults results from a complex interaction among the different cells that form its matrix. The separation of the different epithelial cell types is an essential step in the studies of mechanisms of the controlling factors of cell repair, cell differentiation, and neoplastic transformation. Epithelial cells of the New Zealand white rabbit trachea are prepared using enzymatic digestion and microdissection. Small sections of tracheal wall are dissected into pieces approximately 10 mm2. The mucosa is dissected and placed in 0.15% hyaluronidase for 40 min at 22 degrees C. Mucus is removed, and the mucosa is then placed in 0.1% pronase at 37 degrees C for 30 min. With careful dissection, the epithelium can be dissected from the mucosa in 10-mm2 sheets. Sheets of epithelial cells are placed in 6 ml of an enzymatic solution containing collagenase, 0.2% bovine serum albumin, 0.04% soya bean trypsin inhibitor, 0.06 ml of 1 M Hepes buffer for 3 h at 37 degrees C. The cells are gently pipetted during the 3-h period, yielding a suspension of viable cells. Subpopulations of these different cell types are enriched using an Orthocytofluorograph 50111. A krypton ion laser was used for excitation of cells at 488 nm. Forward-angle and 90 degrees scatter were gated on the histogram. The purification of the ciliated, basal, and secretory cells was 90%, 97%, and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗