title: High-throughput measurement of mitochondrial membrane potential in a neural cell line using a fluorescence plate reader.
abstract: Mutations in mitochondrial genes cause mitochondrial genetic disease, which is often associated with deficiency of the mitochondrial membrane potential (MMP). We present a high-throughput method for measuring MMP in intact neural cells using TMRM, a well-known potentiometric dye, in a 48-well plate format. Addition of known MMP depolarizing agents, FCCP or DNP, resulted in a time- and concentration-dependent decrease in fluorescence, which was saturable, whereas the addition of drugs that affect non-mitochondrial properties did not. A cell line deficient in mtDNA had decreased fluorescence, which was not further depleted by a depolarizing agent. The high-throughput results are similar to those produced by more time-consuming and low-throughput flow cytometry or microscopy methods. This plate-based system could facilitate the identification of cell-permeant small molecules (i.e., drugs) that modify MMP, which could be used to enhance mitochondrial function, and also for screening small populations of neural cells for mutations in nuclear or mtDNA genes that decrease MMP.
authors.affiliation: Department of Molecular Biosciences, University of California, Davis 95616, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Wong
authors.forename: Gino A
authors.initials: GA
authors.lastname: Cortopassi
mesh_terms: D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics; D004140:Dinitrophenols / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D011199:Potentiometry; D012235:Rhodamines
chemical_list: D004272:DNA, Mitochondrial; D004140:Dinitrophenols; D005456:Fluorescent Dyes; D009467:Neuromuscular Depolarizing Agents; D012235:Rhodamines; C401833:tetramethylrhodamine methyl ester; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone