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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↗
PubMed1993

[Present and future of flow cytometry].

Flow cytometry is used for lymphocyte subset identification and leukemia typing. In routine laboratories, leukocyte differentiation and reticulocyte counting are also common uses for flow cytometry. Flow cytometers provide accurate and precise data with simple manipulation. The disadvantage of flow cytometry is the low cost efficiency as a clinical test. Dual color analysis is a recent trend and more information on cells can be obtained, though the selection of antibody subclass sometimes leads to incorrect results. As technology develops, however, flow cytometry becomes more feasible in routine clinical laboratories, and the number of clinical uses will increase.

Blood Cell Count↗
PubMed1997

Assessment of immunotoxicity by multiparameter flow cytometry.

Flow cytometry is a unique technology useful in the examination of effects of immunotoxic agents on target cells of the immune system. The purpose of this workshop was to provide an overview of the use of flow cytometry in new and established models of immunotoxicity, with emphasis on the potential applications, assay validation, and potential pitfalls. This overview begins with a discussion of methods useful in the assessment of Ca2+-dependent mechanisms of lymphoid cell activation in surface marker-defined human B cells, T cells, and monocytes. A discussion of the use of flow cytometry in analysis of apoptosis is also presented in this paper. The second paper presents data on the development and use of flow cytometry as an alternative to a Cr51 release assay for an assessment of cytotoxic T cell activation. The use of surface markers for characterizing and distinguishing the effects of chemical irritants from sensitizers is next presented, followed by an overview of the use of fluorescent probes to assess cell thiol status and overall oxidant-induced injury to lymphoid cells. Finally, an interlaboratory study designed to compare and evaluate the use of flow cytometry procedures in rat splenic cell subtyping is presented. Overall, these studies demonstrate the utility of flow cytometry assays in immunotoxicologic research, but further efforts are needed in the validation of many of these assays for routine use in immunotoxicologic testing.

Allergens↗
PubMed1996

Enumeration and biomass estimation of bacteria in aquifer microcosm studies by flow cytometry.

Flow cytometry was used to enumerate and characterize bacteria from a sand column microcosm simulating aquifer conditions. Pure cultures of a species of Bacillus isolated from subsurface sediments or Bacillus megaterium were first evaluated to identify these organisms' characteristic histograms. Counting was then carried out with samples from the aquifer microcosms. Enumeration by flow cytometry was compared with more-traditional acridine orange direct counting. These two techniques gave statistically similar results. However, counting by flow cytometry, in this case, surveyed a sample size 700 times greater than did acridine orange direct counting (25 (mu)l versus 0.034 (mu)l) and required 1/10 the time (2 h versus 20 h). Flow cytometry was able to distinguish the same species of bacteria grown under different nutrient conditions, and it could distinguish changes in cell growth patterns, specifically single cell growth versus chained cell growth in different regions of an aquifer microcosm. A biomass estimate was calculated by calibrating the total fluorescence of a sample from a pure culture with the dry weight of a freeze-dried volume from the original pure culture. Growth conditions significantly affected histograms and biomass estimates, so the calibration was carried out with cells grown under conditions similar to those in the aquifer microcosm. Costs associated with using flow cytometry were minimal compared with the amount of time saved in counting cells and estimating biomass.

Journal Article↗
PubMed1994

A new combined integral-light and slit-scan data analysis system (DAS) for flow cytometry.

Flow cytometry using list mode parameters such as fluorescence emission, light scatter and size on one hand and different slit-scan parameters on the other hand needs a fast, flexible, efficient and easy-to-use data analysis software. A new software package (data analysis system, DAS) has been developed that integrates data analysis for conventional (integral-light) flow cytometry and for slit-scan flow cytometry. The requirements, design and some examples are discussed and an implementation for IBM-compatible computers is presented. Special attention is directed to the handling of different data types from one-parameter histograms to multiparameter slit-scan data files. The package can be used as an interpreting programming language or as an interactive menu-driven command line interpreter with a large number of graphic, mathematical and statistical functions. DAS is not limited to use in flow cytometry only, but multidimensional data analysis, from astronomy to economics, can be done as well.

Data Display↗
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↗
PubMed2000

Analysis of virus-infected cells by flow cytometry.

Flow cytometry has been used to study virus-cell interactions for many years. This article critically reviews a number of reports on the use of flow cytometry for the detection of virus-infected cells directly in clinical samples and in virus-infected cultured cells. Examples are presented of the use of flow cytometry to screen antiviral drugs against human immunodeficiency virus (HIV), human cytomegalovirus, and herpes simplex viruses (HSV) and to perform drug susceptibility testing for these viruses. The use of reporter genes such as green fluorescent protein incorporated into HIV or HSV or into cells for the detection of the presence of virus, for drug susceptibility assay, and for viral pathogenesis is also covered. Finally, studies on the use of flow cytometry for studying the effect of virus infection on apoptosis and the cell cycle are summarized. It is hoped that this article will give the reader some understanding of the great potential of this technology for studying virus cell interactions.

Apoptosis↗
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↗
PubMed2001

Cell function-based flow cytometry.

Flow cytometry is increasingly used to assess the functional status of leukocytes, and practically all aspects of their life (and death) are accessible to flow cytometric study. Together with familiar features of flow cytometry, such as multiparameter immunophenotyping, cell function-based flow cytometry has provided many new insights into the relationships among lymphocyte cell surface features; intracellular processes, such as cytokine production and protein phosphorylation; and the functional status of lymphocytes in a variety of human diseases. Direct visualization and quantification of antigen-specific T cells using major histocompatibility complex (MHC)-peptide tetramer technology, in combination with functional assays, has provided the means to study specific T-cell subsets of interest. Even early in its development, this technology already has offered a better understanding of basic and clinical immunology and has invited reassessment of several long-standing immunologic concepts. Semin Hematol 38:169-178. This is a US government work. There are no restrictions on its use.

Animals↗
PubMedDate not supplied

A new analytical method for the detection of anti-tumor promoters using flow cytometry.

Flow cytometry analysis was applied to the measurement of Epstein-Barr virus (EBV) induction which is used as a short term assay for anti-tumor promoters. The data obtained by measurement with flow cytometry were parallel with those of the fluorescence microscopic method. Flow cytometry is rapid, quantitative and should be applicable for the EBV activating test.

Anticarcinogenic Agents↗
PubMed1999

Fundamentals of flow cytometry.

Flow cytometers are instruments that are used primarily to measure the physical and biochemical characteristics of biological particles. This technology is used to perform measurements on whole cells as well as prepared cellular constituents, such as nuclei and organelles. Flow cytometers are investigative tools for a broad range of scientific disciplines because they make measurements on thousands of individual cells/particles in a matter of seconds. This is a unique advantage relative to other detection instruments that provide bulk particle measurements. Flow cytometry is a complex and highly technical field; therefore, a basic understanding of the technology is essential for all users. The purpose of this article is to provide fundamental information about the instrumentation used for flow cytometry as well as the methods used to analyze and interpret data. This information will provide a foundation to use flow cytometry effectively as a research tool.

Computers↗
PubMed1996

Ataxonomic assessment of phytoplankton integrity by means of flow cytometry.

Flow cytometry, a method well established in medicine and biotechnology, can also make an important contribution to (applied) limnological as well as ecotoxicological studies on phytoplankton. Flow cytometry can, for instance, contribute to the ataxonomic structural and functional assessment of phytoplankton. This approach may serve as a supplement to the well-established taxonomic evaluation by means of various microscope techniques. We present some examples for such ataxonomic phytoplankton evaluation. These examples include phytoplankton of eutrophicated and acidified water bodies as well as slowly flowing rivers. Phytoplankters may be differentiated by their pigment contents into carotinoid-rich ones (such as Chrysophyceae, Bacillariophyceae, and Dinophyceae) and carotinoid-poor ones (such as Euglenophyceae and Chlorophyceae). As a useful biomass parameter of phytoplankton algae we tested successfully protein staining by fluorescein isothiocyanate. We discuss the advantage of this approach as compared with results obtained by Coulter counter or by biomass calculations from microscope analyses. Up to now, evaluation of the biological quality of pelagic water bodies is still laborious and time consuming because of the microscopical examination of planktic communities usually practiced. As a possible improvement we present a structural ataxonomic approach for assessing the integrity of individual phytoplankters (on the basis of physiological parameters) as well as of the phytoplankton communities that is based on annual means of biomass spectra. Flow cytometry can provide considerable relief.

Biomass↗
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↗
PubMed1994

A phenotypic study of CD8+ lymphocyte subsets in infants using three-color flow cytometry.

Flow cytometry is a powerful tool for the multiparametric evaluation of cell surface phenotype in patients with HIV disease. Many cell surface molecules can be evaluated by three-color flow cytometry and the markers correlated with functional activity. It has recently been recognized in adults that the CD8 cell is an important lymphocyte subset in HIV disease that correlates with disease outcome, but there is little information about CD8 subsets in infants. Therefore, we studied infants born to HIV-infected mothers and those born to uninfected mothers. No significant differences were seen in phenotypic markers of activation (CD38, HLA-DR), maturation (CD45RO, CD45RA), and function (CD28) between uninfected infants born to HIV infected or uninfected mothers. In HIV-infected infants, a substantial increase in CD8+ CD38+ HLA-DR+ expression was seen. In addition, we found that there was a significant increase in the CD8+ CD45RO+ CD45RA- subset which is characteristic of the memory phenotype. Finally, evaluation of CD28 (costimulatory molecule involved in T cell activation), which is expressed on almost all CD8 cells at birth, showed that this population was significantly reduced in infected infants. These studies suggest that three-color flow cytometry is a powerful tool for evaluating phenotypic changes in lymphocyte subsets and enhancing our understanding of the pathobiology of HIV disease.

CD8 Antigens↗
PubMed2005

Monitoring of active but non-culturable bacterial cells by flow cytometry.

Flow cytometric signatures (i.e., light scatter, red and green fluorescence) were obtained for the active but non-culturable (ABNC) cells of E. coli and a coliform isolate H03N1, in seawater microcosms using BacLight, a live-dead assay kit from Molecular Probes (Eugene/Portland, OR). Previous studies have reported that there are two major adaptations, which cells undergo during the formation of ABNC states: cell wall toughening and DNA condensation. Therefore, we hypothesized that the matured ABNC forms should be more resistant to extreme temperature treatments (i.e., by freezing in liquid nitrogen and thawing at room temperature) than the normal and transition populations. It was shown that the membrane-compromised cells (comprising of normal wild-type and dead cells which are less resistant to rapid freeze thaw) could be differentiated from the matured ABNC using BacLight staining and fluorescence detection by flow cytometry. The population of ABNC cells, which could not be cultured using m-FC media (for the enumeration of fecal coliforms), was resuscitated in phosphate buffer saline followed by growth in Luria broth. Flow cytometry was thus able to detect and differentiate the ABNC cells against a mixed population comprising of culturable cells, transition populations, and dead cells. The results also showed that the formation of ABNC is as early as 2 days in seawater microcosms. By directly comparing the coliform levels enumerated by the BacLight based flow cytometry assays and m-FC technique, it was shown that the presence of coliforms can be undetected by the membrane filtration method.

Cell Count↗
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↗