ADAPTATION OF A SIMPLE FLOW CYTOMETRIC ASSAY TO IDENTIFY DIFFERENT STAGES DURING APOPTOSIS

Apoptosis occurs under many different physiological and pathological c onditions, and it reflects a genetically encoded suicide program that can be triggered by different stimuli in susceptible cells. We adapted a now cytometric assay for the detection of apoptosis based on differ ential staining of viable cells with two different DNA binding dyes, p ropidium iodide (PI) and Hoechst 33342 (Ho342). Apoptosis was induced in different cell lines by gamma irradiation, an anti-PAS monoclonal a ntibody, or ganciclovir in Herpes simplex virus-thymidlne kinase-expre ssing cells. We could identify three different populations that appear ed sequentially after the induction of apoptosis. Cells corresponding to these populations were sorted and assessed for evidence of apoptosi s as determined by alterations of nuclear morphology and detection of endonucleolytic activity. This analysis revealed a PI- population with subtle apoptotic changes and increased Ho342 fluorescence compared wi th untreated cells. Extensive apoptotic alterations were observed in a PI+ population that increased over time following the induction of ap optosis. A third population was characterized by an intermediate inten sity of PI fluorescence and decreased Ho342 fluorescence compared with the other populations. This population appeared late after treatment and consisted of apoptotic bodies. Taken together, these data suggest that distinct stages of apoptosis can be identified by differential st aining of cells with Ho342 and PI. This assay should be useful for the detection and further characterization of cells at different stages i n the apoptotic process. (C) 1996 Wiley-Liss, Inc.