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At least 19 recordsLinked to original sources

PubMed1991

Attachment of A172 human glioblastoma cells affects calcium signalling: a comparison of image cytometry, flow cytometry, and spectrofluorometry.

The intracellular free calcium concentration ([Ca2+]i) of indo-1 loaded A172 human glioblastoma cells stimulated by platelet-derived growth factor (PDGF) was studied in cell suspensions by flow cytometry and spectrofluorometry and in confluent monolayers by laser image cytometry and spectrofluorometry. With all three techniques, the percentage of responsive cells, peak [Ca2+]i, and the duration of response were directly related, and the delay time was inversely related to PDGF dose. The maximum response occurred at a PDGF concentration of about 20 ng/ml. Basal and peak [Ca2+]i did not differ significantly from method to method even though different calibration procedures were used. Cells in suspension monitored by both spectrofluorometry and flow cytometry displayed significantly shorter calcium responses than attached cells. This did not appear to be a direct effect of trypsinization. Spectral analysis of indo-1 in cytoplasm, 40% glycerol, and aqueous solutions showed significant differences in the isosbestic point and quantum efficiency. Calibration of [Ca2+]i with spectrofluorometry is more accurate using the ratio of fluorescence intensities than the fluorescence intensities measured at either 405 or 485 nm.

Calcium↗

PubMedDate not supplied

Evaluation of the platelet surface by fluorescence flow cytometry.

Flow cytometry has significantly contributed to cell biology, especially with use of tagged monoclonal antibodies. Discovery of specific platelet surface membrane receptors has helped to elucidate the pathogenesis of several platelet related disorders. Concomitantly, monoclonal antibodies to specific receptors have also been developed. Using fluorescent antibody techniques, small populations of platelets with distinct characteristics can be analyzed efficiently. Monoclonal antibodies, e.g., (AP-3), specific for glycoprotein, IIIa and (6DI), directed against an epitope close to the VWF binding site, have already been employed,--the latter by fluorescence flow cytometry. In addition to studies of primary surface membrane disorders, flow cytometry has been applied to measurements of surface immunoglobulin and C3 on individual platelets in autoimmune and alloimmune thrombocytopenia. Flow cytometry may also be utilized for platelet crossmatching. This simple, specific and non-radioactive technique can effectively provide accurate platelet surface data in a small clinical laboratory.

Antigens, Surface↗

PubMed1988

Quality control in clinical flow cytometry.

Flow cytometry is a multifaced technology which requires attention to the details of sample preparation as well as instrumentation. QC procedures are essential for minimizing technical artifacts which may lead to erroneous results. In clinical laboratories it is particularly important to establish QC procedures that are readily performed with relatively few control reagents. The ultimate goal of these QC procedures is to permit the acquisition of precise and accurate results which will be of importance in clinical diagnoses and patient care. The current technology of commercially available flow cytometry instruments and fluorescent immunologic reagents has indicated that consistent results can be attained with a variety of instruments, reagents, and different flow cytometry operators [10, 20]. Ideally, the formulation of guidelines for quality assurance laboratory practices in flow cytometry will provide a means for all clinical laboratories to obtain accurate and precise results.

Antibodies, Monoclonal↗

PubMed1985

Clonal stability and heterogeneity of hybridomas: analysis by multiparameter flow cytometry.

Flow cytometry of cellular DNA and RNA content was employed to determine DNA ploidy, proliferation, and RNA content of hybridoma cultures shortly after cell fusion and sequentially thereafter. The parental mouse myeloma cell line P3UI was characterized by tetraploid DNA content, S-phase 50%, and high RNA, the parental mouse spleen cells by diploid DNA content, low proliferation, and low RNA content. Hybridoma cultures studied as early as 21 days after fusion were found to contain the sum of the parental cells' DNA content (hexaploid), or if less, more than that of the myeloma parental cells. Only one clone of 35 tested was shown to be hexaploid and the rest hypertetraploid or hyperpentaploid. Hybrid cell cultures were frequently found to contain a variable mixture of unfused parental cells. The high proliferation of hybridoma cells determined by flow cytometry indicates that these cells would eventually overgrow the parental cells. Flow cytometry also enabled an accurate estimation of the effect of various doses of dexamethasone added to HAT medium immediately after cell fusion on hybridoma formation. Cultures treated with 10(-5) mM of the hormone had a higher DNA ploidy than cultures grown in the presence of 10(-3) mM dexamethasone. No parental cells were observed in the hybridoma cultures studied with this hormone. Sequential DNA/RNA measurements of hybridoma clones showed a decrease in DNA ploidy over time with high dexamethasone doses and a minimal increase or no change with low hormone dose. Flow cytometry is suggested to be a useful technique for evaluating the effects of various agents on DNA ploidy and proliferation and on stability of fused cells.

Animals↗

PubMed1985

Monitoring intravesical bacillus Calmette-Guerin treatment of bladder carcinoma with flow cytometry.

Flow cytometry of bladder irrigation specimens was studied in 22 patients with low stage bladder carcinoma who were treated by transurethral resection of visible tumor followed in 3 to 5 weeks by a course of intravesical bacillus Calmette-Guerin. The most informative examinations were just before the first bacillus Calmette-Guerin treatment, 6 weeks after completing a 6-week course of treatment (3 months) and at 9 months. Of the patients 10 had recurrent tumors after therapy; recurrence was anticipated correctly by flow cytometry at the 12-week followup examination in 6 of the 10 patients and suspected in another. Of 12 patients who remained clinically free of disease for a minimum of 15 months after bacillus Calmette-Guerin therapy flow cytometry identified correctly 7 at 12 weeks, while 1 had a partial response and the remaining 4 reverted to a negative status at 9 months. Of interest, only 4 of the 22 patients were free of disease by flow cytometry at the start of bacillus Calmette-Guerin treatment despite attempted ablation of the tumor by transurethral resection, suggesting that intravesical administration of bacillus Calmette-Guerin destroys existing carcinoma in situ in some cases.

BCG Vaccine↗

PubMed1989

Studies of DNA content in cervical intraepithelial neoplasia by cytologic and histologic flow cytometry.

Flow cytometry was done on 82 specimens of cervical cytologic scrapings and biopsies from patients with cervical intraepithelial neoplasia or condyloma. This study yielded significant results when compared with standard cytologic (p less than 0.01) and histopathologic studies (p less than 0.05). Histologic evaluation by flow cytometry required more examinations and did not give significant results. The authors conclude that cytologic flow cytometry, though not a replacement for standard cytologic examination, may be of help in establishing therapeutic strategies and follow-up protocols for patients with cervical precancerous conditions.

Aneuploidy↗

PubMed1986

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry.

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.

Animals↗

PubMed1989

Procedural guidelines for performing immunophenotyping by flow cytometry.

Flow cytometry is a rapidly expanding technology that is moving from the research laboratory into the clinical laboratory. Recent advances in availability and reproducibility of monoclonal antibody reagents specific for a wide range of cell types coupled with lower costs for increasingly automated flow cytometers with powerful and user friendly data analysis capabilities have made flow cytometry the method of choice for immunophenotyping in the clinical laboratory. However, there is great variability in the level and type of quality assurance procedures used from laboratory to laboratory. A subcommittee established by the National Committee for Clinical Laboratory Standards (NCCLS), composed of representatives from industry, academia, professional societies, and regulatory agencies, has drafted consensus procedures which address specific problems and suggested solutions for performance of immunophenotyping by flow cytometry. This paper is based on the authors' discussions with the NCCLS Committee but does not represent an official NCCLS position. The official NCCLS document on this subject (H42) is expected to be published in 1989.

Adult↗

PubMed1991

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.

Animals↗

PubMed1990

Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry.

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.

Chlorophyll↗

PubMedDate not supplied

Analysis of coccidian oocyst populations by means of flow cytometry.

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mitis caused difficulty in separation from the remaining fine debris. This technique was useful for studying the mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Characterization is further limited by the lack of suitable size/shape standards for flow cytometry.

Animals↗

PubMed1990

Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry.

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.

Animals↗

PubMed1992

Detection of borreliacidal antibodies in hamsters by using flow cytometry.

Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.

Animals↗

PubMed1986

Update on the concept of the cell cycle: the contribution of flow cytometry.

Flow cytometry has been extensively used to provide accurate estimates of the relative amounts of various cellular constituents (DNA, RNA, proteins) for cell kinetic studies. Multiparametric analysis also supports the recent concept that cell growth and the DNA division cycle may be under distinct regulatory mechanisms. Moreover, metabolic subcompartments of the cell cycle, distinguished by flow cytometry, have offered a highly sensitive cell classification in comparison with the conventional distinction of the four main phases of the cell cycle. Finally, a new sensitive and powerful technology, BrdU/DNA analysis, represents a remarkable maturing of a very useful alternative for the study of DNA synthesis and cell cycle traverse.

Animals↗

PubMed1992

Estimating percentage constitutive heterochromatin by flow cytometry.

Flow cytometry is a powerful method for the assessment of both plant and animal genomes. One of the most interesting aspects is the analysis of chromatin structure. By using intercalating and base pair-specific fluorochromes, the chromatin structure in various cell cultures and microorganisms has been determined. In this study, several maize lines of known heterochromatic composition were analyzed. The nuclei of each line were isolated and stained with DAPI (base pair specific) and PI (intercalator) separately. For each maize line, the PI/DAPI ratio was determined. A significant negative correlation was observed between C-band number and PI/DAPI ratio (r = 0.920) and between percentage heterochromatin and PI/DAPI ratio (r = 0.997). Flow cytometry with use of the fluorochromes DAPI and PI was found to be a rapid and efficient method of determining heterochromatin amount in maize.

Chromosome Banding↗

PubMed1989

Separation of pigmented and albino melanocytes and the concomitant evaluation of endogenous peroxide content using flow cytometry.

Flow cytometry (FCM) has been used extensively to analyze various biological properties of the cell. In this report, we describe a method by which FCM was used to determine the light scattering profile of a mixed population of pigmented and non-pigmented melanocytes, plus its subsequent use for the sorting and separation of the two cell types. In addition, the relative peroxide content in pigmented and non-pigmented melanocytes was compared by flow cytometry. Cultured avian melanocytes from a pigmented control and from three genetically distinct albino sources were studied. FCM analysis of forward versus side light scatter within a mixed suspension of pigmented and amelanotic melanocytes distinguished two overlapping populations of cells. Sorting of these two populations demonstrated that the population exhibiting much side and minimal forward light scatter was primarily pigmented melanocytes, while conversely the population exhibiting less side and more forward scatter was principally non-pigmented cells. These two melanocyte types also demonstrated differences in levels of endogenous peroxides. The intracellular content of peroxide in the two subpopulations of cells was measured utilizing the nonfluorescent compound, 2',7'-dichlorofluorescein diacetate (DCFH-DA), which within the cell is oxidized by intracellular peroxides to a fluorescent dichlorofluorescein (DCF). Non-pigmented albino melanocytes had the highest quantity of endogenous peroxides, while heavily pigmented cells had considerably less peroxide-related fluorescence. The amount of this DCF fluorescence could be enhanced by increasing concentrations of DCF used in the assay. These flow cytometric methods are useful for isolating and culturing subpopulations of melanocytes expressing various pigment levels and to investigate the relationship between melanin and its precursors with hydrogen and lipid peroxides in melanocytes.

Animals↗

PubMed1988

Primary squamous cell carcinoma of the male urethra: nuclear deoxyribonucleic acid ploidy studied by flow cytometry.

Flow cytometry analysis was performed on 30 primary male urethral squamous cell carcinoma specimens. Nuclei were extracted from paraffin-embedded archival material and isolated nuclei were stained with propidium iodide. Bulbomembranous urethral tumors had a higher incidence of abnormal deoxyribonucleic acid ploidy patterns than penile urethral tumors (69 and 29 per cent, respectively). Of the tumors exhibiting a deoxyribonucleic acid diploid pattern and an abnormal (deoxyribonucleic acid tetraploid or aneuploid) histogram 18 and 93 per cent, respectively, showed tumor progression (p less than 0.001). None (0 per cent) of the low grade (grade 1 or 2) tumors with a deoxyribonucleic acid diploid pattern developed local recurrence or distant metastases, whereas 90 per cent of the low grade tumors with an abnormal deoxyribonucleic acid pattern progressed (p less than 0.002). Patients with tumors exhibiting deoxyribonucleic acid diploid ploidy had 5 and 10-year rates free of disease of 85 per cent. In contrast, patients with tumors with abnormal deoxyribonucleic acid ploidy patterns had 5 and 10-year rates of 20 and 0 per cent, respectively (p less than 0.001). Determination of deoxyribonucleic acid ploidy pattern by flow cytometry provides important prognostic information for male patients with primary squamous cell carcinoma of the urethra.

Adult↗

PubMed1988

In vivo expression of rubella antigens on human leucocytes: detection by flow cytometry.

Flow cytometry has been used to detect in vivo expression of rubella antigens on human leucocytes. Sequential samples of peripheral blood were obtained from four volunteers with naturally acquired rubella and five persons immunised with RA27/3 rubella vaccine. Leucocytes were stained for rubella antigens using a pool of rubella monoclonal antibodies. Rubella antigens were detected on the leucocytes of all four volunteers with naturally acquired rubella between 1 and 13 days after onset of illness. Viral antigens were expressed more frequently on the monocyte (9-51%) than the lymphocyte (less than 1-4%) and granulocyte (less than 1-3%) populations. Among the vaccines, rubella antigens were detected on the leucocytes of four of the five volunteers between 5 and 12 days after immunisation. The expression of viral antigens was more transient and the proportion of cells exhibiting rubella-specific fluorescence considerably lower following vaccination (1-12%) than natural infection (9-51%). Our results demonstrate that flow cytometry provides a rapid and sensitive analytical technique for detecting viral antigens on leucocytes from infected persons. Leucocytes may play an important role in the pathogenesis of rubella infection.

Adult↗