THE USE OF FLOW-CYTOMETRY FOR THE INVESTIGATION OF CELL-DEATH

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying b y apoptosis. There are different interpretations of changes observed i n DNA histograms of these cells. We describe an approach based on extr action of chromatin degradation products from fixed cells and subseque nt staining with DNA specific dyes. Apoptotic cells containing fragmen ted DNA are observed in <2C DNA region of DNA histograms. DNA histogra ms of irradiated thymocytes dying in vitro and stained without extract ion of fragmented DNA do not differ from control. Under the same stain ing conditions DNA histograms of lymphocytes dying in thymus of irradi ated animals reveal fluorescent material in <2C DNA region, most likel y due to formation of apoptotic bodies (cell fragments, some of them c ontain fragments of nuclei). Similar changes are observed in thymocyte s dying upon glucocorticoid treatment. Our present results and other d ata indicate that reduced amount of DNA in dying cells is the main rea son for changes of DNA histograms. Examples of application of the meth od described for the investigations of cell death modifiers are presen ted. (C) 1993 Wiley-Liss, Inc.