Cytosolic acidification and mitochondrial dysfunction during apoptosis as probed by dual emission probes and microspectrofluorometry

Cytosolic acidification and mitochondrial dysfunction have been reported to be concomitant with apoptosis. In our study, we report on the use of carboxy-SNARF-1-AM and JC1 probes in association with confocal scanning microspectrofluorometry to examine respectively cytosolic pH and mitochondrial membrane potential during camptothecin (CPT) and daunorubicin (DNR) induced apoptosis in human leukemic HL60 cells. Emission intensity ratios (I_{580 nm}/I_{640 nm}) of carboxy-SNARF-1 spectra lead to determine cytosolic pH. Whereas, JC1 was able to form J-agregates respectively from green (530 nm) to yellow-orange (590 nm) as mitochondrial membrane becomes more polarised. Our results show that control cells presented a neutral cytosolic pH (7.26 ± 0.08). The cells induced in apoptosis in presence of 1 μM CPT or DNR during 6hrs, undergo significant acidification of the cytosol (7.00 ± 0.06 and 7.02 ± 0.05 respectively). Decrease of I_{590 nm}/I_{530 nm} emission ratio of JC1 spectra (64 ± 5 % compared to control) was observed only with CPT. We conclude that cytosolic acidification and mitochondrial dysfunction are early events in apoptosis and may be essential for genome destruction. Confocal scanning microspectrofluorometry and dual ratiometric fluorescent probes appear to be a powerful approach for the study of ion response in living cells.