Flow cytometric detection of apoptosis: Comparison of the assays of in situ DNA degradation and chromatin changes

Abstract

The aim of this study was to compare three methods of detection of apoptotic cells: (1) the method based on elution of low molecular weight DNA from the ethanol fixed cells followed by cell staining with DAPI (diamidino‐2–phenylindole) or propidium iodide as the DNA fluorochromes, (2) the method of in situ labeling of DNA strand breaks with biotinylated dUTP, utilizing exogenous terminal deoxyribonucleotide transferase, and (3) the method of analysis of DNA denaturation in situ using acridine orange to differentially stain denatured and doublestranded DNA sections following cell exposure to 0.1 M HCl. Cells of the human promyelocytic HL‐60 line, treated in vitro with the DNA topoisomerase I inhibitor camptothecin, which selectively triggers apoptosis of S‐phase cells, were chosen as a model. The method based on analysis of changes in DNA denaturability was the most sensitive in terms of detection of the earliest changes in chromatin of cells undergoing apoptosis; the increased sensitivity of DNA to denaturation in Sphase cells was measured as early as 100 min after addition of camptothecin. DNA cleavage, assayed either by the univariate measurement of DNA content following extraction of low molecular weight DNA, or by labeling DNA strand breaks with biotinylated dUTP, was detected in S‐phase cells after 120 min incubation with camptothecin. The percentage of apoptotic cells at the late stage of apoptosis, the kinetics of cell transition to apoptosis, and kinetics of the loss of S phase cells were all essentially similar when measured by any method. All three methods can be used to estimate the cell cycle phase specificity of apoptosis, although the method based on DNA strand labeling with biotinylated dUTP by terminal deoxynucleotidyl transferase has the advantage of making it possible to estimate the cell cycle distribution of both the apoptotic and unaffected cell populations. The latter method also appears to be the most specific in terms of detection of apoptosis. © 1994 Wiley‐Liss, Inc.

Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells

“…Cells (10 6 ) were washed in phosphate buffered saline (PBS), fixed in 70% ethanol, incubated with 100 mg ml 71 RNase A and 40 mg ml 71 propidium iodide (PI) (Sigma, UK) in citrate-buffered saline for 30 min at 378C (Hotz et al, 1994). DNA histograms were generated by fluorescence activated cell sorting (FACS) analysis using an EPICS Elite ESP cell sorter (Beckman-Coulter, High Wycombe, UK) at 488 nm.…”

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia

“…Assays currently used to detect apoptosis include detection of morphologic changes by light microscopy, electron microscopy or flow cytometry using nuclear staining, intensity of fluorescence, cell size, identification of a sub-G 1 peak on DNA ploidy analysis and FITC-labelled annexin V (Nicoletti et al, 1991;Sun et al, 1992;Hotz et al, 1994;Koopman et al, 1994). Internucleosomal cleavage products produced by the endonucleases are also used to detect apoptosis.…”

“…An increase was also demonstrated in the proportion of S phase cells just before undergoing apoptosis, followed by the appearance of a sub-G 1 peak representing the apoptotic cells and loss of S phase cells at a later stage (Nicoletti et al, 1991;Hotz et al, 1994).…”

“…In situ labelling of DNA strand breaks with labelled dUTP and ddUTP appears to be a very specific method of detecting apoptosis (Gorgczyca et al, 1994;Hotz et al, 1994;Gavrieli et al, 1992;Schmitz et al, 1991). In situ labelling has been shown to be more specific when using TdT (terminal deoxynucleotidyl transferase) instead of DNA polymerase and when digoxigenin-labelled nucleotides rather than the biotin:streptavidin system are used (Ben-Sasson et al, 1995;Schmitz et al, 1991).…”

Transient receptor potential melastatin type 7 channels are involved in zinc-induced apoptosis in gastric cancer

“…As a method to analyze the mode of cell death in AGS cells by zinc, we used sub-G1 analysis (Hotz et al 1994;Vermes et al 2000). In this protocol, AGS cells with zinc are stained with a fluorescent DNA stain (such as PI).…”