FLOW CYTOMETRIC ANALYSIS OF MITOCHONDRIAL ACTIVITY IN-SITU - APPLICATION TO ACETYLCERAMIDE-INDUCED MITOCHONDRIAL SWELLING AND APOPTOSIS

Changes in mitochondrial matrix volume were studied both on isolated m itochondria and in situ on CHME 5 human microglia and monoblastoid U 9 37 cells using multiparametric flow cytometric analysis, The use of sp ecific effecters of mitochondrial activity (oligomycin and KCN) allowe d the demonstration, on whole cells, of a strict correlation between l ight scattering and mitochondrial volume changes: mitochondrial swelli ng induced a concomitant increase in forward scattering, and decrease in side scattering of the cell population. The technique was applied t o the study of the early phases of acetyl-ceramide-induced apoptosis, which has been associated with mitochondrial dysfunction in several ce llular systems. Acetyl-ceramide caused a marked swelling of isolated r at liver mitochondria. Scatter modifications were also observed in bot h cell lines during the first hour of incubation with acetylceramide a nd were accompanied by an increase in DiOC(6) (3) fluorescence. The re sults imply that mitochondrial volume changes can be followed using fl ow cytometry and eventually used to assist in the interpretation of mi tochondrial membrane potential variations obtained from fluorescence m easurements. By applying this technique to 2 different cell lines, we demonstrated that mitochondrial swelling occurs during the early phase s of acetyl-ceramide treatment, but that the induction of apoptosis is cell type-dependent, Cytometry 33:333-339, 1998, (C) 1998 Wiley-Liss, Inc.