A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes

Sun, Xiaoming; Snowden, Roger T.; Skilleter, David N.; Dinsdale, David; Ormerod, M.G.; Cohen, Gerald M.

doi:10.1016/0003-2697(92)90251-2

Cited by 129 publications

(91 citation statements)

References 15 publications

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“…Sequential cell staining with Hoechst 33342 and PI was used by Sun et al,(62) and Ormerod et al,(63) to discriminate live, apoptotic and dead cells, the latter having a permeable plasma membrane.…”

Section: Note Added In Proofsmentioning

confidence: 99%

Features of apoptotic cells measured by flow cytometry

et al. 1992

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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 Sphase 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 apoptotic 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 oaf the apoptotic process. (b) Plasma membrane integrity, which is lost in necrotic but not in 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 shatter, 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 love 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. The data presented indicate that flow cytometry can be applied in basic research on molecular and biochemical mechanisms of apoptosis, as well as in the clinic, where the ability to monitor early signs of apoptosis in samples from patients' tumors may be predictive of the outcome of some treatment protocols. © 1992 Wiley‐Liss, Inc.

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Section: Note Added In Proofsmentioning

confidence: 99%

Features of apoptotic cells measured by flow cytometry

et al. 1992

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“…We have previously described a flow cytometric method for the separation and quantitation of viable normal and apoptotic thymocytes [30]. The basis of this method appears to be the increased permeability of the apoptotic compared to the normal cells [31].…”

Section: Apoptosis Assessed By Flow Cytometrymentioning

confidence: 99%

An ICE‐like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells

1995

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“…Subsequent to treatment, thymocytes were incubated with Hoechst 33342 and propidium iodide to quantify the percentage of apoptotic cells, essentially as previously described [22], except that analysis was performed using the Becton Dickinson FACS Vantage flow cytometer. The cells with high blue fluorescence due to increased Hoechst 33342 intensity have previously been shown to be apoptotic based on a number of criteria including decreased size, ultrastructure and the presence of DNA ladders [22,23].…”

Section: Quantitation Of Apoptosis By Flow Cytometrymentioning

confidence: 99%

“…The cells with high blue fluorescence due to increased Hoechst 33342 intensity have previously been shown to be apoptotic based on a number of criteria including decreased size, ultrastructure and the presence of DNA ladders [22,23].…”

Section: Quantitation Of Apoptosis By Flow Cytometrymentioning

confidence: 99%

A role for metals and free radicals in the induction of apoptosis in thymocytes

1994

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Recent reports have implicated a possible but undefined role for reactive oxygen species in the induction and mediation of apoptosis. In the present study, the role of free radicals and metal ions in apoptosis induced in rat thymocytes by dexamethasone and etoposide was examined. Copper chelators, but not iron specific chelators, inhibited apoptosis induced by both these stimuli. Several antioxidants also possessed potent inhibitory effects. We therefore propose that diverse agents may induce apoptosis in thymocytes by a common mechanism involving a copper mediated Fenton reaction, generating site specific hydroxyl radicals, possibly as a result of activation of the redox sensitive transcription factor NF-KB.

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A flow-cytometric method for the separation and quantitation of normal and apoptotic thymocytes

Cited by 129 publications

References 15 publications

Features of apoptotic cells measured by flow cytometry

Features of apoptotic cells measured by flow cytometry

An ICE‐like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells

A role for metals and free radicals in the induction of apoptosis in thymocytes

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