JC-1, but not DiOC6(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 cytofluorimetric analysis of mitochondrial membrane potential (delta psi) 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, DiOC6(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 DiOC6(3). Second, a comparative study was performed concerning the effects of agents capable of collapsing deltapsi. While adding FCCP to cell cultures resulted in consistent changes in the fluorescence emission of both JC-1 and DiOC6(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 delta psi 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 [DiOC6(3)]. These data cast some 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 apoptosis.
Salvioli
S
S
Department of Biomedical Sciences, Section of General Pathology, Modena, Italy.
Ardizzoni
A
A
Franceschi
C
C
Cossarizza
A
A
D017209:Apoptosis; D001562:Benzimidazoles / Q000493:pharmacokinetics; D002232:Carbocyanines / Q000493:pharmacokinetics; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D007425:Intracellular Membranes; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D015203:Reproducibility of Results; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured; D014475:Uncoupling Agents
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007476:Ionophores; D012235:Rhodamines; D014475:Uncoupling Agents; D020112:Rhodamine 123; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D011189:Potassium Chloride