Background Information

Key events taking place during apoptosis include loss of mitochondrial membrane potential, decrease in mitochondrial electron transport, oxidation of mitochondrial cardiolipin, and altered cellular redox status (Green and Kroemer, 1998; Green and Reed, 1998). After staining cells with fluorescent dyes such as JC-1, CMXRosamine, and DiOC6(3), changes in mitochondrial parameters were demonstrated by flow cytometry (Cossarizza et al., 1994; Macho et al., 1996; Backway et al., 1997). Initially it was not clear whether the changes observed by flow cytometry were independent of each other or components of one or several pathways of apoptosis. By staining cells simultaneously with dyes for several biochemical parameters that exhibit distinct fluo rescence emission ranges, Backway et al. (1997) demonstrated in leukemic blast cells that loss of mitochondrial membrane potential took place after glutathione was depleted. This loss of mitochondrial membrane potential preceded intracellular formation of reactive oxygen. By performing multiparameter flow cy-tometry, the authors were able to map a "death sequence." Poot and Pierce (1999) further expanded this concept and showed that camp-tothecin treatment of human lymphoblastoid cells led to the formation of a cell subpopulation that underwent a simultaneous decrease in mi-tochondrial membrane potential, NADH level, and oxidative turnover.

Cells stained with NAO show strong fluorescence in the green, yellow, and red regions of the spectrum; therefore, NAO cannot be

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