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Results for “Flow Cytometry”

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PubMed1983

Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry.

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.

Antigens, Bacterial↗
PubMed1977

Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes.

Requirements for flow cytometry of metaphase chromosomes stained with three deoxyribonucleic acid (DNA)-specific fluorescent dyes--Hoechst 33258, Chromomycin A3, and ethidium bromide--are reviewed. Fluorescence properties of these three stains when bound to mitotic cells or to chromosomes in suspension are measured and compared with fluorescence properties when bound to DNA in solution. Conditions are given for high resolution flow cytometry of Chinese hamster chromosomes stained with each of the fluorophors, and histograms are presented that exhibit differences in relative peak position and area. Energy transfer fluorescence between two DNA stains is presented as a potentially useful new parameter for flow cytometry of chromosomes and is illustrated by fluorescence energy transfer from Chromomycin A3 to ethidium bromide when simultaneously bound to hamster mitotic cells.

Animals↗
PubMed2005

Monitoring population dynamics of the thermophilic Bacillus licheniformis CCMI 1034 in batch and continuous cultures using multi-parameter flow cytometry.

Multi-parameter flow cytometry was used to monitor the population dynamics of Bacillus licheniformis continuous cultivations and the physiological responses to a starvation period and a glucose pulse. Using a mixture of two specific fluorescent stains, DiOC6(3) (3,3'-dihexylocarbocyanine iodide), and PI (propidium iodide), flow cytometric analysis revealed cell physiological heterogeneity. Four sub-populations of cells could be easily identified based on their differential fluorescent staining, these correspond to healthy cells (A) stained with DiOC6(3); cells or spores with a depolarised cytoplasmic membrane (B), no staining; cells with a permeabilised depolarised cytoplasmic membrane (C), stained with PI; and permeablised cells with a disrupted cytoplasmic membrane 'ghost cells' (D), stained with both DiOC6(3) and PI. Transmission electron micrographs of cells starved of energy showed different cell lysis process stages, highlighting 'ghost cells' which were associated with the double stained sub-population. It was shown, at the individual cell level, that there was a progressive inherent fluctuation in physiological heterogeneity in response to changing environmental conditions. All four sub-populations were shown to be present during glucose-limited continuous cultures, revealing a higher physiological stress level when compared with a glucose pulsed batch. A starvation period (batch without additional nutrients) increased the number of cells in certain sub-populations (cells with depolarised cytoplasmic membranes and cells with permeabilised depolarised cytoplasmic membranes), indicating that such stress may be caused by glucose limitation. Such information could be used to enhance process efficiency.

Algorithms↗
PubMed1992

Analysis of lymphocyte activation and metabolism by flow cytometry.

The use of flow cytometry for the study of lymphocyte activation and cellular metabolism continues to expand. This has permitted rapid progress in the understanding of the regulation of multiple intracellular ions, metabolic pathways, and the control of gene expression. Knowledge of these basic mechanisms of cellular homeostasis is now being applied to the flow cytometric study of immune dysregulation. Various flow cytometric techniques for measuring apoptosis are summarized.

Animals↗
PubMed2000

Screening of new antioxidant molecules using flow cytometry.

We present a flow cytometry technique to evaluate the antioxidative properties of molecules on living cells, using a stable murine-murine hybridoma (Mark 3) cell line routinely cultured. Using this technique, intracellular superoxide anions and peroxides were evaluated with dihydrorhodamine (DHR-123) and dichlorofluorescein diacetate (DCFH-DA), respectively. When cells were first incubated for 10 min with either H(2)O(2) or the xanthine (X)/xanthine oxidase (XO) system, this flow cytometric technique was capable of evaluating the oxidative stress on cells. Twenty-one new analogues of ellipticine were synthesized and tested for their antioxidative properties compared to vitamin E and Ebselen used as references. A good statistical reflection of the antioxidative activities of these molecules was achieved by analyzing 35 000 cells in each experiment. Among them, the selenated molecule 18 was found to be 10 times more active than Ebselen but 10 000 times less active than vitamin E. Moreover, eight compounds showed glutathione peroxidase-like activities.

Animals↗
PubMed2004

Flow cytometry: an introduction.

A flow cytometer is an instrument that illuminates cells (or other particles) as they flow individually in front of a light source and then detects and correlates the signals from those cells that result from the illumination. In this chapter, each of the aspects of that definition will be described: the characteristics of cells suitable for flow cytometry, methods to illuminate cells, the use of fluidics to guide the cells individually past the illuminating beam, the types of signals emitted by the cells and the detection of those signals, the conversion of light signals to digital data, and the use of computers to correlate and analyze the data after they are stored in a data file. The final section of the chapter will discuss the use of a flow cytometer to sort cells. This chapter can be read as a brief, self-contained survey. It can also be read as a gateway with signposts into the field. Other chapters in this book will provide more details, more references, and even some controversy about specific topics.

Animals↗
PubMed2004

[Applications of flow cytometry in toxicological test].

Flow cytometry (FC) gained attention as a powerful high-throughput and multiparameter technology for the analysis of molecular and cell events in biological testing. With the developing of the instrument capability and the improving of the detection methods. FC has been becoming a more important tool both for research and clinical testing practice. FC has also been used in many toxicological experiments, which greatly promotes the progress of toxicological techniques. In this paper, the recent progress on the flow cytometric approach used in toxicological detection was reviewed.

Animals↗
PubMed1992

[Applications of flow cytometry to molecular biology and molecular genetics].

Flow cytometry has been utilized in the fields of immunology, hematology, cancer biology and somatic cell genetics. It is also being used for cell cycle and chromosome ploidy analysis. Furthermore, flow cytometry is powerful for karyotype analysis and chromosome sorting. Here, I will review recent advances of flow cytometry in molecular biology and molecular genetics, and discuss some of our recent works.

Animals↗
PubMed1999

[Laser spatial scanning in flow cytometry].

The main flow laser cytometry principles, based on the elastic light scattering, spheres of its applications, problems of its realization and utilization in the immunological investigations and diagnostics are analysed. The experimental model of a flow cytometer with laser probing beam space scanning, originally proposed by the authors, is described. The apparatus was tested by polystyrene latex spheres and biological objects. The experiments showed that the achieved sensitivity was enough to register red blood cells, their complexes and bacterial cells.

Flow Cytometry↗
PubMed2004

Application of flow cytometry to platelet disorders.

Flow cytometry is a powerful and versatile tool that can be used to yield definitive information regarding the phenotypic status of platelets. The method provides a quantitative assessment of the physical and antigenic properties of platelets (e.g., surface expression of receptors, bound ligands, components of granules, or interactions of platelets with other platelets, other blood cells, or components of the plasma coagulation system), thereby facilitating the diagnosis of inherited or acquired platelet disorders (e.g., Bernard-Soulier syndrome, Glanzmann thrombasthenia, storage pool disease), the pathological activation of platelets (e.g., in the setting of acute coronary syndromes, cerebrovascular ischemia, peripheral vascular disease, cardiopulmonary bypass), and changes in the ability of platelets to activate via specific stimuli (e.g., efficacy of antiplatelet therapies). Accordingly, this review summarizes the key technical and methodologic components of flow cytometric analysis of platelets, as well as specific examples of its application to diagnosis and patient care.

Blood Platelet Disorders↗
PubMedDate not supplied

Quantifying heterogeneity: flow cytometry of bacterial cultures.

Flow cytometry is a technique which permits the characterisation of individual cells in populations, in terms of distributions in their properties such as DNA content, protein content, viability, enzyme activities and so on. We review the technique, and some of its recent applications to microbiological problems. It is concluded that cellular heterogeneity, in both batch and continuous axenic cultures, is far greater than is normally assumed. This has important implications for the quantitative analysis of microbial processes.

Bacteria↗
PubMed2005

Monitoring and quantification of inclusion body formation in Escherichia coli by multi-parameter flow cytometry.

Multi-parameter flow cytometry was used to monitor the formation of promegapoietin (PMP) inclusion bodies during a high cell density Escherichia coli fed-batch fermentation process. Inclusion bodies were labelled with a primary antibody and then with a secondary fluorescent antibody. Using this method it was possible to detect PMP inclusion body formation with a high specificity and it was possible to monitor the increased accumulation of the protein with process time (6-48 mg PMP/g CDW) whilst highlighting population heterogeneity.

Bioreactors↗
PubMed1987

Quantitative assessment of neutrophil function by flow cytometry.

The use of flow cytometry (FCM) to quantitatively assess neutrophil function is reviewed. The methodology is capable of measuring a number of parameters involved in the oxidative pathways that form the basis of the activated neutrophil's contribution to the host defense mechanism. These events are summarized and some findings, such as in patients with chronic granulomatous disease, are discussed. FCM study of neutrophil function requires smaller numbers of cells than do traditional methods, which makes it particularly useful in the assessment of small fluid samples or in the evaluation of multiple parameters, and has the advantage that cell purification procedures are not essential.

Flow Cytometry↗
PubMed2003

The origin and identification of unknown events associated with low-level leucocyte counting by flow cytometry.

BACKGROUND AND OBJECTIVES: Flow cytometric enumeration of residual leucocytes (WBC) in leucocyte-depleted components occasionally reveals a population of events that stain with propidium iodide, but which are outside the main counting region. These extraregional events may result in discrepancies in residual WBC counts. MATERIALS AND METHODS: To identify the origin of these unknown events, separate populations of mononuclear cells and polymorphonuclear neutrophils (PMNs) (prepared by density-gradient centrifugation) were spiked into leucocyte-depleted red cell concentrates (RCC) to a concentration of 150 cells/ microl and assessed, using LeucoCount and DNA Prep reagents, by flow cytometry. In addition, isolated WBC nuclei, DNA in saline, or enzymatically digested free DNA, was spiked into diluted whole blood (final concentration 5 microg/ml). RESULTS: Isolated WBC nuclei fell in the main counting region of the dot plot. Extraregional events were generated in leucocyte-depleted RCC spiked with PMN (17/ microl by day 4), but not with mononuclear cells, and increased with sample storage (4 days). A significantly (P < 0.01) greater number of extraregional events was observed using the LeucoCount reagent, compared with the DNA Prep reagent. Enzymatically digested WBC nuclei or free DNA, added to diluted whole blood, generated extraregional events using LeucoCount, but not the DNA Prep reagent. When free DNA was enzymatically digested, no extraregional events were observed. CONCLUSIONS: These previously unidentified events are probably fragmented nuclei or free DNA originating from PMN owing to a combination of ageing and reagent addition. Currently, in our protocols, the region used for WBC enumeration counts intact WBC nuclei. To achieve WBC counting consistency, flow cytometric gating protocols must be standardized, and a decision taken as to whether to include extraregional events in the count.

Cell Nucleus↗
PubMed1992

Analysis and isolation of renal tubular cells by flow cytometry.

The cells of the renal cortex have rich heterogeneity of structure and function. Flow cytometry, the technique of rapid laser-based single cell analysis, can give information about cellular mixtures not obtainable by any other means. We examined a variety of fluorescent markers to identify populations of renal cells by flow cytometry. Cellular digests of rat cortex were fluorescently stained with either enzymatic activity probes, or polyclonal antibodies. Fluorescent staining for the proximal marker gamma-glutamyl-transpeptidase (tau-GT) was an order of magnitude brighter than autofluorescence, and stained 71 +/- 11% of the cells. Second, we colocalized enzymatic and antibody markers. There was tight colocalization of tau-GT enzyme activity, detected with fluorogenic substrates, with specific surface binding of tau-GT antibodies. Third, populations of fluorescently labelled cells can be rapidly isolated by flow cytometry sorting. Flow cytometry sorting isolated 10(7) cells positive for the proximal tubular marker tau-GT in a little under one hour. The sorted cells were viable with 99 +/- 2% trypan blue exclusion (N = 8). Sodium-dependent phloridzin-inhibitable glucose uptake was present in sorted cells, with greater uptake/mg protein than in unsorted controls. The sorted cells grew in culture as a monolayer of tightly adherent cuboidal cells. Hence, flow cytometry allows us to quantitate the heterogeneity in mixed renal cellular digests. Flow cytometry allows us to rapidly isolate millions of cells according to fluorescently tagged markers. The isolated cells are viable, retain sodium-dependent transport properties, and grow in culture.

Alkaline Phosphatase↗
PubMed1994

Detection of Salmonella typhimurium in dairy products with flow cytometry and monoclonal antibodies.

Flow cytometry, combined with fluorescently labelled monoclonal antibodies, offers advantages of speed and sensitivity for the detection of specific pathogenic bacteria in foods. We investigated the detection of Salmonella typhimurium in eggs and milk. Using a sample clearing procedure, we determined that the detection limit was on the order of 10(3) cells per ml after a total analysis time of 40 min. After 6 h of nonselective enrichment, the detection limits were 10 cells per ml for milk and 1 cell per ml for eggs, even in the presence of a 10,000-fold excess of Escherichia coli cells.

Animals↗