Journal• ISSN: 0196-4763
Cytometry
Wiley
About: Cytometry is an academic journal. The journal publishes majorly in the area(s): Flow cytometry & Population. It has an ISSN identifier of 0196-4763. Over the lifetime, 2876 publications have been published receiving 121979 citations.
Topics: Flow cytometry, Population, Propidium iodide, Cytometry, Cell cycle
Papers published on a yearly basis
Papers
TL;DR: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis, applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomersase inhibitors or prednisolone.
Abstract: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
2,038 citations
TL;DR: The basic mechanisms underlying the loss of membrane asymmetry during apoptosis are described and the novel annexin V-binding assay is discussed, an extension to the current available methods.
Abstract: Apoptosis is a programmed, physiological mode of cell death that plays an important role in tissue homeostasis. Understanding of the basic mechanisms that underlie apoptosis will point to potentially new targets of therapeutic treatment of diseases that show an imbalance between cell proliferation and cell loss. In order to conduct such research, techniques and tools to reliably identify and enumerate death by apoptosis are essential. This review focuses on a novel technique to detect apoptosis by targeting for the loss of phospholipid asymmetry of the plasma membrane. It was recently shown that loss of plasma membrane asymmetry is an early event in apoptosis, independent of the cell type, resulting in the exposure of phosphatidylserine (PS) residues at the outer plasma membrane leaflet. Annexin V was shown to interact strongly and specifically with PS and can be used to detect apoptosis by targeting for the loss of plasma membrane asymmetry. Labeled annexin V can be applied both in flow cytometry and in light microscopy in both vital and fixed material by using appropriate protocols. The annexin V method is an extension to the current available methods. This review describes the basic mechanisms underlying the loss of membrane asymmetry during apoptosis and discusses the novel annexin V-binding assay. Cytometry 31:1–9, 1998. © 1998 Wiley-Liss, Inc.
2,011 citations
TL;DR: A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested and makes this system an ideal complement to flow cytometry.
Abstract: A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested Cells stained sequentially with biotinylated antibodies, fluorochrome-conjugated avidin, and superparamagnetic biotinylated-microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns Unlabelled cells pass through the column, while labelled cells are retained The retained cells can be easily eluted More than 10(9) cells can be processed in about 15 min Enrichment rates of more than 100-fold and depletion rates of several 1,000-fold can be achieved The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry Light scatter and fluorescent parameters of the cells are not changed by the bound particles Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence-activated cell sorting without further treatment Magnetic tagging and separation does not affect cell viability and proliferation
1,852 citations
TL;DR: The new detergent technique for the preparation of nuclei for flow cytometric DNA analysis seems well suited as a routine clinical procedure and no cell loss was caused by storage or staining.
Abstract: A new modification of our detergent technique for the preparation of nuclei for flow cytometric DNA analysis is described. The attainment of low coefficients of variation of the peaks and of quantitative staining of nuclei from different tissues was a problem with the original method. This was solved in the new modification by trypsinization of the unfixed nuclei. The nuclei were stabilized by spermine. A simple procedure for long-term storage of samples at —80°C was integrated into the method. The fluorescence of the nuclei was stable for at least 3 hours after staining. Light exposure protection of the samples was essential. No cell loss was caused by storage or staining. The method was successfully applied on samples including: (a) Normal tissues— human lymphocytes, granulocytes and spleen. Mouse lymphocytes, bone marrow, spleen, liver, kidney and thymus. (b) Human neoplasms— lung cancer, breast cancer, lymphoma, leukemia, bladder cancer and cancer of the oral cavity. (c) Human tumors in nude mice— breast cancer, lung cancer, melanoma and colon cancer. (d) Mouse ascites tumors— JB-1, L 1210, Ehrlich and P 383. It therefore seems well suited as a routine clinical procedure.
1,733 citations
TL;DR: A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations and could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells.
Abstract: A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.
1,290 citations
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Performance Metrics
| Year | Papers |
|---|---|
| 2009 | 1 |
| 2007 | 2 |
| 2005 | 33 |
| 2004 | 4 |
| 2003 | 2 |
| 2002 | 133 |