A New Method for the Cytofluorometric Analysis of Mitochondrial Membrane Potential Using the J-Aggregate Forming Lipophilic Cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1)

Abstract

A new method for the cytofluorimetric analysis of mitochondrial membrane potential in intact cells has been developed by using the lipophilic cationic probe 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1), whose monomer emits at 527 nm after excitation at 490 nm. Depending on the membrane potential, JC-1 is able of forming J-aggregates that are associated with a large shift in emission (590 nm). The color of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. In two human cell lines (K562 and U937), we have studied by flow cytometry the changes in membrane potential provoked by the K+ ionophor valinomycin, a drug known to affect mitochondrial membrane potential, while the K+/H+ ionophor nigericin, known to affect intracellular pH but not mitochondrial membrane potential, was used as control. The incubation with valinomycin for 10 min. at 37°C in a low K+ medium provoked a marked and dose-dependent reduction in JC-1 greenish orange fluorescence, while nigericin had no effect.