A fast kinetic method for assessing mitochondrial membrane potential in isolated hepatocytes with rhodamine 123 and flow cytometry.
Rhodamine 123 (Rh123) is widely used as a flow cytometric probe for mitochondrial membrane potential (MMP) in metabolic, pharmacologic, and toxicological studies. However, the use of relatively high concentrations of Rh123 (up to 10 micrograms/ml) and prolonged incubation times (up to 1 h), including washing steps, may be inconvenient for certain applications in which labile cells are used or which demand rapid or repeated analysis. In this paper we describe a rapid kinetic assay of MMP in isolated rat hepatocytes, based upon the quantitation of the initial rate of Rh123 uptake by living cells, selected by their scattering properties. The results indicate that at an appropriate dye-to-cell ratio (in our experiments, 50 ng Rh123/ml for 250,000-300,000 cells/ml), the initial rate of Rh123 uptake is a highly reproducible and sensitive parameter for estimation of MMP, as demonstrated by the effects of substrates and inhibitors of the glycolytic pathway and mitochondrial respiration. Because of its simplicity, rapidity (about 5 min) and metabolic implications, this assay would be also suitable for the routine evaluation of metabolic state of cell suspensions, as a complementary test to the standard dual-staining tests of viability. Other possible applications in screening pharmacologic and toxicological analysis are discussed.
Departamento de Bioquímica, Facultad de Medicina, Universidad de Valencia, Spain.
G
G
Juan
M
M
Cavazzoni
G T
GT
Sáez
J E
JE
O'Connor
D000818:Animals; D001692:Biological Transport; D002470:Cell Survival; D004734:Energy Metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology*; D007700:Kinetics; D008027:Light; D008297:Male; D008564:Membrane Potentials; D008930:Mitochondria, Liver / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism; D012542:Scattering, Radiation
D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123; D005947:Glucose