Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis

Abstract

Apoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent-based lysing buffer increased the fluorescence of toxicant-induced apoptotic nuclei to the level of untreated diploid nuclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation-induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16-h culture, we obAnalysis of the DNA content of thymocyte nuclei prepared by detergent-mediated cytolysis has become a common method for single-parameter flow cytometric detection and quantitation of apoptotic cells (6,8,10, 15,21.24,37). On a DNA histogram, apoptotic nuclei appear as a subdiploid peak clearly distinguishable from the Go&, peak. The mechanism underlying the appearance o f the subdiploid peak has been hypothesized as either decreased fluorochrome binding due to chromatin condensation (6,33) or actual loss of DNA either while the cells are in culture or during sample preparation (2,8,17).In this paper, we report that the addition of a specific RNase A preparation to the lysing buffer resulted in a dramatic increase in the DNA fluorescence of toxicantinduced apoptotic nuclei such that they could not be discriminated from nonapoptotic nuclei. We also were able to mimic this effect by adding 2 mM CaCIJMgCI, to the lysing buffer. Subsequent analysis of the DNA content o f the lysate indicated that the upscale fluorescence shift was the result of a decreased ability of the detergentbased lysing buffer to extract DNA, most likely as a consequence of cation-induced changes in chromatin conformation (9,18,20,3 I ) In addition, we demonstrate that served a similar, but time-dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 p M tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 p M dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. Key terms: Apoptosis, stainability, thymocyte, chromatin, ribonuclease during a 16-h exposure, thymocytes induced to undergo apoptosis either spontaneously or by treatment with 1 pM tributyltin methoxide (TBT), 2% ethanol, 2% me...

Cytotoxicity of tributyltin in rat hippocampal slice cultures

“…Because this is one of the typical characteristics of apoptosis (Walker et al, 1988;Elstein and Zucker, 1994;Elstein et al, 1995), TBT-induced cell death might be due to an apoptosis-like process. The slow time course of TBT neurotoxicity (\ 12 h) also suggests apoptotic cell death.…”

“…condensation is a typical characteristic in apoptosis (Walker et al, 1988;Elstein and Zucker, 1994;Elstein et al, 1995), we investigated the effect of cycloheximide and actinomycin D, widely used as apoptosis inhibitors (Walker et al, 1988;Maurer and Wray, 1997a,b;BenAri and Khrestchatisky, 1998), on TBT neurotoxicity in order to determine whether TBT-induced cell death was apoptotic. However, they alone caused severe neurodegeneration in our hippocampal slice cultures, even at low concentrations (3.5 mM cycloheximide and 4 mM actinomycin D), and so we failed to verify the possibility.…”

Effect of themicrobial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

“…Cells were analyzed using a FACScan (Becton Dickinson, USA) with Cell Fit software. Cells that have lost DNA due to the apoptosis will take up less stain and will appear to the left of the G1 peak (sub-G1) (Gorczyca et al, 1993;Gong et al, 1994;Elstein et al, 1995).…”

Image cytometry of early nuclear events during apoptosis induced by camptothecin in HL-60 leukemic cells

“…For instance, the addition of divalent cations or polyamines like spermine*+ prevents both the opening of the chromatin structure and the induction of apoptosis (4,34). These cations, which increase chromatin compactness ( l ) , could reduce DNA accessibility and thereby reduce the susceptibility of DNA to nuclease digestion (12). These data thus suggested that apoptosis could proceed through endonuclease activation followed by DNA degradation, which would imply a primary opening of the chromatin structure.…”

“…However, this appears unlikely because the presence of cells with an apparently decondensed chromatin was only transient and disappeared after 3 h when typical apoptotic cells could be detected. Conversely, this phenotype could represent early changes associated with apoptosis because the chromatin of cells undergoing apoptosis typically exhibits large breaks and then decondensation and margination as endonucleases degrade internucleosomal linker sections of DNA (2,12,27,33). The induction of apoptosis by CAM in HL-60 cells has been shown to result in very rapid responses, particularly a transient increase of cyclin BlKdc2 kinase activity (31).…”