JC-1, BUT NOT DIOC(6)(3) OR RHODAMINE-123, IS A RELIABLE FLUORESCENT-PROBE TO ASSESS DELTA-PSI CHANGES IN INTACT-CELLS - IMPLICATIONS FOR STUDIES ON MITOCHONDRIAL FUNCTIONALITY DURING APOPTOSIS

The sensitivity and specificity of three fluorescent probes used for c ytofluorimetric analysis of mitochondrial membrane potential (Delta Ps i) were studied in the U937 human cell line. First, the role of plasma membrane in influencing the binding of the probes to mitochondria has been investigated, The depolarization of plasmamembrane with high dose s of extracellular KCI had no immediate effects on the loading of JC-1 , DiOC(6)(3) and rhodamine 123 (R123), However, after a few hours of c ulture in the presence of KCI, significant changes were observed only in cells stained with DiOC(6)(3), Second, a comparative study was perf ormed concerning the effects of agents capable of collapsing Delta Psi , While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC(6)(3) - but not of R1 23 - only cells stained with JC-1 responded to valinomycin. On the who le, our data indicate that JC-1 is a reliable probe for analyzing Delt a Psi changes with flow cytometry, while the others show a lower sensi tivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC(6)(3)]. These data cast s ome doubts on those studies that, using fluorescent probes that have a low sensitivity to Delta Psi hypothesized that the fall in Delta Psi is one of the early events, if not one of the main causes, of apoptosi s. (C) 1997 Federation of European Biochemical Societies.