Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells.
Mitochondrial membrane potential, in situ, is an important indicator of mitochondrial function and dysfunction. Because of recent interest in the role of mitochondria in signaling, cell injury and cell death, there is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Deltapsim, in situ, in a heterogeneous cell population. We have adapted a flow cytometry method for the quantitative measurement of DeltaPsim which utilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)). We developed a new protocol in which cells are equilibrated with very low dye concentrations (<1 nM). Only under these condition, the cell fluorescence appears to be correlated with the magnitude of DeltaPsim, as evident from the sensitivity of the fluorescence to low concentrations of uncouplers, ionophores and inhibitors of the mitochondrial proton pumps. The magnitude of the plasma membrane potential, DeltaPsip, also affects cell fluorescence, and a procedure that corrects for this effect is outlined. This method offers a distinct advantage over existing methods for estimation of Deltapsim by flow cytometry.
Rottenberg
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H
Pathology Department, m.s. 435, Allegheny University of the Health Sciences, MCP/Hahnemann School of Medicine, Broad and Vine streets, Philadelphia, PA 19102, USA.
Wu
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S
D000818:Animals; D002232:Carbocyanines; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002452:Cell Count; D019069:Cell Respiration / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000502:physiology; D008214:Lymphocytes / Q000502:physiology* / Q000648:ultrastructure; D008564:Membrane Potentials; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000502:physiology*; D009840:Oligomycins / Q000494:pharmacology; D011189:Potassium Chloride; D054328:Proton Pump Inhibitors; D012402:Rotenone / Q000494:pharmacology; D013154:Spleen / Q000166:cytology; D014634:Valinomycin / Q000494:pharmacology
D002232:Carbocyanines; D005456:Fluorescent Dyes; D009840:Oligomycins; D054328:Proton Pump Inhibitors; D012402:Rotenone; D014634:Valinomycin; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D011189:Potassium Chloride