DETECTION OF APOPTOSIS BY FLOW-CYTOMETRY OF CELLS SIMULTANEOUSLY STAINED FOR INTRACELLULAR PH (CARBOXY SNARF-1) AND MEMBRANE-PERMEABILITY (HOECHST-33342)

Intracellular acidification appears to be a frequent and possibly univ ersal occurrence during apoptosis. We have used carboxy SNARF-1 to mea sure intracellular pH by how cytometry, We report here that the additi on of Hoechst 33342 concurrently with carboxy SNARF-1 provides a clear er discrimination of the apoptotic population, Apoptotic cells accumul ate Hoechst 33342 more rapidly than do viable nonapoptotic cells, The cells with greater Hoechst 33342 fluorescence are the same cells as th ose with decreased intracellular pH; these cells also have decreased v olume, The different parameters in this analysis are presented for thr ee models of apoptosis: human myeloblastoid ML-1 cells incubated with etoposide, murine cytotoxic T cells following withdrawal of interleuki n-2, and Chinese hamster ovary cells incubated with staurosporine, In most circumstances, carboxy SNARF-1 provides excellent resolution of v iable and apoptotic cells, However, the addition of Hoechst 33342 is p articularly valuable when carboxy SNARF-1 alone can not fully discrimi nate the two cell populations, This situation occurs in Chinese hamste r ovary cells, which undergo a smaller degree of acidification than do the other cell models, This situation also occurs when the extracellu lar pH is experimentally reduced to investigate the mechanism of pH dy sregulation; the apoptotic cells appear to retain the ability to regul ate intracellular pH at low pH, although they are defective in proton export at neutral pH. (C) 1996 Wiley-Liss, Inc.