The use of flow cytometry for the investigation of cell death

Afanasyev, V. N.; Korol, B.A.; Matylevich, Natalya P.; Pechatnikov, V A; Umansky, Samuil R.

doi:10.1002/cyto.990140604

Cited by 61 publications

(31 citation statements)

References 17 publications

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“…The validity of this model for tumor cells in vivo is supported by the similarities of cellular responses (decreased forward and increased 90" light scatter, membrane permeabilization, viability probe uptake, decreased DNA stainability, etc.) with those observed in other tumor model systems in which hypoxia has been shown to induce both necrosis and apoptosis (1,2,7,10,12,15,16,20,22,33). This application imposes highly stringent criteria on a viability probe.…”

Section: Discussionmentioning

confidence: 78%

“…Following incubation with the viability probes, the cells (except for PI-stained controls) were processed according to our solid tumor antibody staining procedure to determine the best probe for this application. The viability probes evaluated in study 1 included PI, antiactin indirectly labelled with sheep-a-mouse-fluorescein isothiocyanate (SAM-FITC), anticytokeratin-SAM-FITC, 7-aminoactinomycin D (7-AAD; a high-affinity DNA intercalator; 24,25), and TO-PRO-3 (a membrane-impermeant, high-affinity DNA intercalator). Viability probes evaluated in study 2 included PI, antitubulin-SAM-FITC, ethidium monoazide bromide (EMA; a photoactivated, covalently binding DNA intercalator; 23), and laser dye styryl (LDS)-751 (a vital nucleic acid stain; 29).…”

mentioning

confidence: 99%

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Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry

O’Brien¹,

Bolton²

1995

Cytometry

120

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Dead cells represent a si@cant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining.To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells Grom each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-a-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specitic staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufticiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its speciticity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe. o 1995 Wiley-Liss, hc.

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Section: Discussionmentioning

confidence: 78%

mentioning

confidence: 99%

Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry

O’Brien¹,

Bolton²

1995

Cytometry

120

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“…DNA degradation is a widely used hallmark of apoptosis and, thus, apoptotic cells can be measured by flow cytometry by determining the proportion of cells that contain less than 2C DNA (Afanasyev et al, 1986(Afanasyev et al, , 1993. Figure 1 shows that about 34% of dead non-adherent cells contain less than 2C DNA and are presumed to be apoptotic based on this criterion.…”

Section: Resultsmentioning

confidence: 99%

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

et al. 1997

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Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its antiapoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

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“…Irradiated PBMC were double stained with EB and mitochondria transmembrane potential dependent (Rh123) and independent probe (NAO). NAO and Rh123 uptake increased simultaneously in EB-cells, likely reflecting very early phenomena that occur in the mitochondria membrane assembly and activity during apoptosis ( 3 1 ). There was a major difference, however, between the behavior of Rh123 and NAO uptake.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques

et al. 1996

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The early events occurring during apoptosis at the plasma membrane, chromatin, and mitochondrial levels were investigated in freshly isolated irradiated human lymphocytes, growth factor‐deprived cultured human lymphocytes, and dexamethasone (DEX)‐treated murine thymocytes. In intact, unfixed cells, evaluation of the light scatter properties and of DNA stainability with ethidium bromide (EB) allowed a cell subset suggestive for initial apoptosis to be identified. The apoptotic nature of these cells was confirmed by cell sorting in irradiated human lymphocyte model. EB could not be substituted for by propidium iodide, indicating that the nature of DNA probe used is of major importance for detecting initial apoptotic changes. Because mitochondria are thought to represent a primary target during apoptosis, we measured the uptake of mitochondria transmembrane potential sensitive (Rhodamine 123) and nonsensitive (10‐nonyl‐acridine‐orange) probes concomitantly with EB uptake. Cells starting apoptosis had an enhanced incorporation of both mitochondria dyes, which in combination with EB identified several cell subsets. This suggests that complex alterations in mitochondrial structure and functioning occur in the early stages of apoptosis. To investigate phenomena occurring at the chromatin level in similar phases of apoptosis, irradiated human lymphocytes and DEX‐treated murine thymocytes were disrupted and DNA stainability assessed in nuclear suspensions. A transient increase in DNA stainability, i.e., the appearance of distinct hyperdiploid peaks in the human model and a generalised upward shift of the G0/1 peak in the murine model, was observed in the early phases of apoptosis concomitantly with specific alterations in light scattering properties. These findings suggest that chromatin texture is altered in early apoptosis and affects DNA stainability. © 1996 Wiley‐Liss, Inc.

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The use of flow cytometry for the investigation of cell death

Cited by 61 publications

References 17 publications

Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry

Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion

Flow cytometric analysis of the early phases of apoptosis by cellular and nuclear techniques

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