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Results for “Flow Cytometry”

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PubMed1987

Comparative analysis of monoclonal antibody-drug conjugate binding by flow cytometry.

Flow cytometric methods for the evaluation of the cell surface binding properties of monoclonal antibody (MoAb)-drug/toxin conjugates defining tumor-associated antigens are presented. In these techniques, suspension cultures of solid human tumor cell lines are incubated with either varying dilutions of MoAb or MoAb-drug conjugates followed by FITC-conjugated anti-mouse immunoglobulin antibodies in an indirect assay or with FITC-conjugated MoAbs specific for the tumor target cell line in a competition assay. The amount of fluorescent probe bound is measured by flow cytometry and the mean fluorescence intensity determined. The relative binding capacity is quantified by linear regression of the mean fluorescence versus the concentration of primary antibody or antibody conjugate. The application of these techniques to several drug and toxin conjugates of MoAb KS1/4, which defines a human adenocarcinoma-associated antigen, demonstrates that these assays can be effectively utilized to monitor the effects of covalent chemical modification on a MoAb's antigen binding reactivity.

Adenocarcinoma↗

PubMed1989

Estimation of DNA content in uveal melanomas by flow cytometry.

Flow cytometry was used to evaluate ploidy and tumour cycle kinetics in fresh tissue samples obtained from 19 uveal melanomas. The results were compared with other parameters including, histological cell type, tumour size and anatomical location. Three tumours (15.8%) were aneuploid (two mixed cell, one epithelioid cell). Cell turnover was estimated in the 16 diploid tumours by summating the total percentage of cells in S and G2/M phases. We found the mean percentage of cells in G2/M/S to be 5.96% (range 2.2-9.8%). Spindle cell neoplasms appeared to have lower cell turnover rates (4.5 +/- 1.2%) than epithelioid cell turnover (8.4 +/- 1.2%). There was no correlation between cell turnover and either tumour size or anatomical location.

Aged↗

PubMed1983

Uptake of latex particles by macrophages: characterization using flow cytometry.

Flow cytometry can be used to characterize the uptake of particles by small samples of pulmonary macrophages both quickly and accurately. We found that there was a linear relationship between the number of fluorescent latex particles and the fluorescent intensity associated with each cell up to 47 particles/cell. Macrophages lavaged from the lungs of Syrian golden hamsters were metabolically and functionally stable for 3 h of incubation; the average intracellular concentrations of Na+ and K+ were 29 +/- 2 and 158 +/- 6 mM, respectively. Particle uptake was significantly inhibited by removing divalent cations with ethylenediaminetetraacetic acid and lowering the incubation temperature (37 degrees C) to 24 and 3 degrees C. The cell-to-cell variability in the uptake of particles was greater than the Poisson distribution would predict based on the mean number of particles per cell.

Animals↗

PubMed1989

Use of lipophilic fluorescent probes for the isolation of hybrid cells in flow cytometry.

Flow cytometry was used for the isolation of hybrid cells immediately after fusion. Precursor cells were stained by two lipophilic fluorescent probes: perylenoyl-labeled triglyceride (perylenoyl-TG, green fluorescence, 520 nm) and rhomdaminyl-labeled triglyceride (rhodaminyl-TG, red fluorescence, greater than 580 nm). Since the maximum emission of perylenoyl-TG coincides with the maximum absorbance of rhodaminyl-TG, the two fluorescent dyes form an effective donor-acceptor pair. Cells stained by perylenoyl-TG (0.25-1 microgram/ml) at the excitation wavelength of 457 nm displayed high intensity of fluorescence in the green region (520 nm), and low intensity of fluorescence in the red region (greater than 580 nm). Using the same conditions, cells that were stained by rhodaminyl-TG displayed a low intensity of fluorescence in both regions. When cells were simultaneously labeled by perylenoyl-TG and rhodaminyl-TG (used in a concentration ratio of 1:10, respectively) essentially total energy transfer was observed, and the cells exhibited a high intensity of red fluorescence. After the fusion of cells which had been separated stained by perylenoyl-TG and rhodaminyl-TG, the hybrid cells containing the two fluorescent probes had a high intensity of red fluorescence. Resonance exitation energy transfer between the two fluorescent dyes permits effective sorting of hybrid cells by flow cytofluorometry.

Cell Fusion↗

PubMed1995

Polymorphonuclear leucocyte dysregulation in patients with gram-negative septicaemia assessed by flow cytometry.

Flow cytometry was used to study phagocytic function and release of reactive oxygen intermediates (ROI) following phagocytosis by granulocytes in 14 patients (six female, eight male) with gram-negative septicaemia prior to, during, and after therapy compared with a group of healthy controls. Phagocytic capacity was assessed by measuring uptake of fluorescein isothiocyanate (FITC)-labelled bacteria. Reactive oxygen generation after phagocytosis was measured by the quantification of dihydrorhodamine 123 converted to rhodamine 123 intracellulary. Compared with results in healthy controls granulocytes of septicaemic patients exhibited a decreased capacity to phagocytize Escherichia coli and to generate reactive oxygen products. Both phagocytosis and ROI production increased after initiation of therapy and normalized within 7 days of treatment. The results suggest that granulocytes do not only participate in, but are also a target of, the septic host inflammatory response.

Adolescent↗

PubMed1999

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Animals↗

PubMed1993

Endocytosis of beta 2 integrins by stimulated human neutrophils analyzed by flow cytometry.

Flow cytometry and fluorescently labeled monoclonal antibodies were used to investigate endocytosis of human neutrophil beta 2 integrins following cellular activation. CD18 initially present on the cell surface cycled in two phases after exposure to formyl peptide or platelet-activating factor. The first phase lasted 3 min at 37 degrees C; after a lag, CD18 was specifically internalized at approximately 20%/min. Subsequently a second phase was detectable consisting of exponential reduction of internal fluorescence with a half-time of approximately 2 min, representing probe reexpression. At peak endocytosis approximately 40% of CD18 was internalized. All of the internalized CD18 was associated with alpha M (CR3); no endocytosis of alpha L (LFA-1) was observed. When neutrophils were stimulated with phorbol esters or calcium ionophore, CD18 was internalized much more slowly (t1/2 = 5 min) and probe was not reexpressed. Endocytosis of CD18 may participate in regulating neutrophil adhesiveness, removing activated receptors, or permitting receptor recycling.

Antibodies, Monoclonal↗

PubMed1992

[Analysis of DNA of renal cell carcinomas by flow cytometry].

Flow cytometry DNA analysis of fragments of fresh or paraffin-embedded tumor was performed in order to investigate its prognostic value in renal cell carcinoma. As aneuploidy is a frequent cytogenetic finding of these cancers, it is not surprising that the literature reports a high incidence of anomalies of DNA content, affecting 2/3 of cases studied. DNA aneuploidy is more frequent in advanced stages, high grades and spindle-cell carcinomas. Its prognostic value in terms of the survival of nephrectomized patients has been demonstrated by unifactorial studies. Several studies using Cox's model have demonstrated its independence of stage and grade, but they are too limited for DNA ploidy to be considered to be an element of the therapeutic decision. In contrast with other sites, the prognostic influence of the proliferation evaluated by S% does not appear to be as important as DNA ploidy. Prospective studies comparing these results should take into account the tumor heterogeneity observed in this type of cancer by analysing, for example, several fragments of the same tumor.

Biopsy↗

PubMed1995

[A new mode of expression for the assessment of capacities of DNA repair by flow cytometry].

Flow cytometry technic was used to study DNA synthesis of Hep G2 cells following mitomycin C and adriblastine treatments during 24 hours. DNA synthesis was expressed by 2 methods: the new expression global DNA synthesis (S+G2)/G1 that considered the cells during scheduled and unscheduled DNA syntheses of S and G2 phases and the cell cycle (Fox program) that evaluated the cells during scheduled DNA synthesis by the terms G1 = 2n, S = 2n+x and G2 = 4n which excluded unscheduled DNA synthesis. The experimental data treated with this new expression led to the determination of threshold concentrations for the two tested compounds where the DNA repair mechanisms were overloaded, leading to cell death. This term was shown to be more accurate to describe the genotoxic action of compounds. Furthermore, these threshold concentrations of DNA damages was found to be linked with significant increase of micronuclei in the micronucleus test.

Antibiotics, Antineoplastic↗

PubMed1988

Uterine leiomyosarcomas and benign smooth muscle tumors: usefulness of nuclear DNA patterns studied by flow cytometry.

Flow cytometry was used to determine the DNA ploidy pattern of paraffin-embedded archival tissue specimens from 90 surgically resected uterine smooth muscle tumors (49 leiomyosarcomas and 41 leiomyomas). The technique of Hedley was used for preparation of paraffin-embedded tissue into single dissociated nuclei, and the method of Vindeløv was used for staining with propidium iodide. Among the 41 leiomyomas, most tumors (88%) had a DNA diploid pattern; the exceptions were two DNA tetraploid/polyploid and three DNA aneuploid samples. The DNA histograms of the 49 leiomyosarcomas (including 6 epithelioid leiomyosarcomas) were classified as follows: 9 cases (18%) exhibited a DNA diploid pattern, 29 cases (59%) had a DNA tetraploid/polyploid pattern, and 11 cases (23%) had DNA aneuploid peaks. Although DNA ploidy pattern cannot be used diagnostically to distinguish malignant from benign uterine smooth muscle tumors, the nuclear DNA ploidy pattern is an easily measured, objective determination that may have important prognostic significance for patients with uterine leiomyosarcomas.

Adult↗

PubMed1998

Indications for a cost-saving policy in routine flow cytometry.

Flow cytometry has known a growing popularity in last decade as an intriguing research and diagnostic tool, favoured by the availability of newly developed monoclonal antibodies directed against cell surface antigens. Unfortunately, equipment and reagents costs are still prohibitive, and it does not allow a more widespread use of this technology. We realised that most producers suggest to add the same amount of reagents irrespective of the real number of target cells, and this is particularly true if the lysed whole blood technique is used. We investigated if an "individualized" concentration of reagents, based on the real number of MNC to be stained, could have allowed us to achieve qualitative satisfactory results with a concomitant reduction in costs. We studied four groups of individuals: 259 healthy subjects (239 blood donors and 20 volunteers) and 10 HIV+ patients. Our results suggest that lower amounts of reagents could be used, at least in some routinary cytometric analysis, simply following providers instructions (i.e. 1 microL of monoclonal antibody/1 x 10(5) MNC).

Adult↗

PubMed1991

Use of quality control standards in clinical flow cytometry.

Flow cytometry is important in the clinical laboratory, especially in hematology and cancer diagnosis. With newer applications being continuously developed clinicians who will use the results and laboratory staff who produce them should be aware of the instrumental quality control procedures required. This will allow evaluation of the reliability as well as the comparison of results between different laboratories. While information about sample preparation as well as staining procedures is easily available, information about instrumental quality control procedures is less so. Various standards and controls are available commercially and their correct choice and usage requires an understanding of the methodology of instrumental quality control. Methods to evaluate the precision, sensitivity and accuracy of measurements are discussed. Most laboratories report immunophenotyping data as the percentage of antibody positive cells and make no attempt to quantitate the amount of antibody binding per cell. Methods for quantitating the number of bound antibody molecules per cell along with a simple technique to compare results between laboratories are discussed. These techniques should encourage laboratory staff and clinicians to standardise methodology and to exchange patient data between laboratories.

Flow Cytometry↗

PubMed1983

Lymphocyte subpopulations in chronic lymphocytic leukemia detected by lectin binding and flow cytometry.

Flow cytometry was used to compare binding of wheat germ agglutinin (WGA) and Lens culinaris lectin (Lcl) to peripheral lymphocytes (PBL) from normal subjects and 26 patients with chronic lymphocytic leukemia (CLL). As defined by the interactions of WGA and Lcl with cell surface receptors, normal unfractionated PBL and purified B-cells were heterogeneous, containing subclasses that bound to WGA (WGA+Lcl-), Lcl (WGA-Lcl+), both (WGA+Lcl+), or neither (WGA-Lcl-). By contrast, in 25 of 26 CLL patients, PBL were homogeneous with approximately 90% of cells binding one or both lectins. Apparent monoclonal tumor cell populations were identified in these patients corresponding to the normal lectin defined cell subclasses: WGA-Lcl- in 18 patients; WGA-Lcl+ in four patients; WGA+Lcl+ in three patients. Ten patients were studied sequentially on more than one occasion at intervals of five to 15 months. Lectin binding patterns remained stable within this time frame. These findings demonstrate that lectin binding may detect several CLL subclasses corresponding to lectin-defined subsets of normal PBL. Lectins can serve as useful tools for dissecting the heterogeneity of human lymphoproliferative diseases.

Flow Cytometry↗

PubMed2005

Assessment of neutrophil activation in whole blood by flow cytometry.

Flow cytometry methods currently used for measuring neutrophil activation involve sample manipulation, which may result in cellular depletion and artifactual activation. To design a new methodology for measurement of neutrophil activation with minimal sample manipulation. Oxidative burst and CD 11b neutrophil expression were simultaneously assessed by a new no-lyse no-wash technique and a standard lyse-method in 10 pediatric patients with recurrent infections and two patients with chronic granulomatous disease (CGD). The new technique was based on nucleic acid staining to discriminate erythrocytes and debris without requiring physical separation. Both methods served equally to confirm or eliminate the diagnosis of CGD and leukocyte adhesion deficiency type 1. The values of baseline CD11b and oxidative burst obtained using the lysis method were significantly higher than those obtained by the no-lyse no-wash method. After activation, the lysis method resulted in higher neutrophil depletion (41%vs. 19%, P = 0.03). When compared with standard methods, neutrophil activation assessment by a no-lyse no-wash method resulted in lower neutrophil depletion and differences in oxidative burst and CD11b neutrophil values.

CD11b Antigen↗

PubMed2006

Data standards for flow cytometry.

Flow cytometry (FCM) is an analytical tool widely used for cancer and HIV/AIDS research, and treatment, stem cell manipulation and detecting microorganisms in environmental samples. Current data standards do not capture the full scope of FCM experiments and there is a demand for software tools that can assist in the exploration and analysis of large FCM datasets. We are implementing a standardized approach to capturing, analyzing, and disseminating FCM data that will facilitate both more complex analyses and analysis of datasets that could not previously be efficiently studied. Initial work has focused on developing a community-based guideline for recording and reporting the details of FCM experiments. Open source software tools that implement this standard are being created, with an emphasis on facilitating reproducible and extensible data analyses. As well, tools for electronic collaboration will assist the integrated access and comprehension of experiments to empower users to collaborate on FCM analyses. This coordinated, joint development of bioinformatics standards and software tools for FCM data analysis has the potential to greatly facilitate both basic and clinical research--impacting a notably diverse range of medical and environmental research areas.

Cell Separation↗

PubMed1998

Effect of cryopreservation of bovine sperm organelle function and viability as determined by flow cytometry.

Flow cytometry was used to compare the functional status of fluorescently stained sperm organelles from 12 Holstein bulls after storage for 24 h at 5 degrees C and after cryopreservation. The organelle-specific stains, SYBR-14 and LysoTracker Green DND-26, identified spermatozoa with intact plasmalemma and those with intact acrosomes, respectively. The mitochondria-specific stain, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1), identified two populations of spermatozoa. One population stained red-orange because the JC-1 accumulated in the mitochondria as aggregates (characteristic of cells exhibiting a high membrane potential); a second population stained green because of JC-1 monomers within the mitochondria (characteristic of cells exhibiting a lower membrane potential). Analysis of variance revealed that within bulls, the properties of sperm viability, intact acrosomes, and mitochondrial status differed in spermatozoa stored for 24 h (p < 0.001) but not in cryopreserved spermatozoa (p > 0.11). Linear regression analyses resulted in significant models in which the proportions of stained spermatozoa stored for 24 h were indicative of those proportions observed in the cryopreserved fractions. These findings suggest that the plasmalemma, the acrosome, and the mitochondria of unfrozen spermatozoa varied as to their functional status. The cryopreservation process, however, resulted in a more uniform status of sperm organelles.

Animals↗

PubMed1992

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.

Animals↗

PubMed2006

Analysis of subcellular sized particles. Capillary electrophoresis with post-column laser-induced fluorescence detection versus flow cytometry.

Flow cytometry (FCM) and more recently capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) have both been used for subcellular particle analysis but their analytical performance has not been compared. In this work, we compare a commercial FCM with an in-house built CE-LIF instrument using fluorescently labeled microspheres and isolated mitochondria. As evidenced by the relative standard deviation (RSD) of the individual fluorescence intensities, FCM is two-fold better than CE-LIF for microspheres with > or =1.5 x 10(6) molecules of equivalent soluble fluorescein (MESF). However, FCM has a comparatively low signal-to-noise ratio (S/N) and high RSD for microspheres with <1.5 x 10(6) MESF. CE-LIF, on the other hand, produces S/N ratios that are >25 times higher than FCM for all the microspheres tested and a lower RSD for microspheres with <1.5 x 10(6) MESF. When 10-N-nonyl acridine orange (NAO)-labeled mitochondria are analyzed, the S/N ratios of both techniques are similar. This appears to result from photobleaching of NAO-labeled mitochondria as they are detected by the LIF detector of the CE-LIF instrument. Both techniques have a niche in subcellular analysis; FCM has the advantage of collecting data for thousands of particles quickly, whereas CE-LIF consumes less than a nanoliter of sample and provides the electrophoretic mobility for individual particles.

Aminoacridines↗