JC-1, but not DiOC 6(3) or rhodamine 123, is a reliable fluorescent probe to assess Δ Ψ changes in intact cells: implications for studies on mitochondrial functionality during apoptosis

Abstract

The sensitivity and specificity of three fluorescent probes used for cytofluorimetric analysis of mitochondrial membrane potential (Δ Ψ) were studied in the U937 human cell line. First, the role of plasmamembrane in influencing the binding of the probes to mitochondria has been investigated. The depolarization of plasmamembrane with high doses of extracellular KCl had no immediate effects on the loading of JC-1, DiOC 6(3) and rhodamine 123 (R123). However, after a few hours of culture in the presence of KCl, significant changes were observed only in cells stained with DiOC 6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing Δ Ψ. 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 R123 – only cells stained with JC-1 responded to valinomycin. On the whole, our data indicate that JC-1 is a reliable probe for analyzing Δ Ψ changes with flow cytometry, while the others show a lower sensitivity (R123), or a non-coherent behaviour, due to a high sensitivity to changes in plasmamembrane potential [DiOC 6(3)]. These data cast some doubts on those studies that, using fluorescent probes that have a low sensitivity to Δ Ψ, hypothesized that the fall in Δ Ψ is one of the early events, if not one of the main causes, of apoptosis.