Sensitive and reliable JC-1 and TOTO-3 double staining to assess mitochondrial transmembrane potential and plasma membrane integrity:: Interest for cell death investigations

Background: Apoptosis is currently studied by flow cytometry with mitochondrial membrane potential (Deltapsi(mt)) and membrane integrity fluorochromes. Rhodamine 123 and DiOC(6)(3) remain controversial to identify cells displaying a low 4-t- JC-1 constitutes a good Deltapsi(mt) indicator, due to a fluorescence shift from green to orange emission, according to the increase in A Deltapsi(mt). Nevertheless, it is not feasible to analyze it simultaneously with propidium iodide. Among available fluorescent probes, TOTO-3 seems to be a good candidate for double staining with JG-1. Methods: Cell death of HaCaT cells was induced by H2O2 and FasL. Samples were stained with DiOC(6)(3)/IP or JC-1/TOTO-3 then analyzed by flow cytometry. Results were supported by confocal microscopy analyses of mitochondrial membrane potential. Moreover, cell morphology was determined on the sorted subpopulations defined on the basis of staining (JC-1 versus TOTO-3). Results: We found that JC-1 is a more efficient mitochondrial probe than DiOC(6)(3). After stress induction, the fluorescence level of JC-1 and TOTO-3 clearly defined three fluorescent subpopulations, respectively: (1) JC-1(high) and TOTO-3(low), (2) JC-1(low) and TOTO-3(medium), and (3) JC-1(low) and TOTO-3(high). Their morphologic aspects after cell sorting indicated that they corresponded to three functional states (intact, apoptotic, and necrotic cells), and data were supported by caspase activity measurements. Conclusions: We propose a reliable and efficient staining, with JC-1 and TOTO-3 to discriminate three functional cellular states: intact, apoptotic, and necrotic/late apoptotic cells by flow cytometry. (C) 2003 Wiley-Liss, Inc.