A FLOW CYTOMETRIC METHOD USING HOECHST-33342 AND PROPIDIUM IODIDE FORSIMULTANEOUS CELL-CYCLE ANALYSIS AND APOPTOSIS DETERMINATION IN UNFIXED CELLS

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensat ion with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it wa s possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apopt otic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured p olymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cel l sorting of the presumed apoptotic population, followed by morphologi c examination or DNA analysis of the sorted populations. The usefulnes s of such a method is discussed in terms of applications in the analys is of heterogeneous clinical samples, populations with low DNA degrada tion during apoptosis, and cell cycle position of the apoptotic cell. (C) 1994 Wiley-Liss, Inc.