We developed a simple ELISA technique as a quantitative in vitro assay for mitophagy in post mitotic skeletal muscle cells. We labeled the mitochondrial DNA of C2C12 myotubes cells by allowing incorporation of the thymidine analogue bromodeoxyuridine (BrdU) and following the decay of the label with time under basal and stimulated conditions. BrdU was incorporated into differentiated C2C12 myotubes at significant levels only after pharmacological treatment was used to increase the rate of mtDNA synthesis. PGCla transduction, stimulation of AMPK, or inhibition of ERK1/2, all previously reported to increase the rate of mtDNA synthesis, induced BrdU incorporation at 4-10 fold over background during a 2 hour incorporation period. Using AMPK stimulation to stimulate initial DNA labeling we confirmed by both microscopy and cytometry methods that BrdU was incorporated into mitochondria and not into nuclei. After removal of the BrdU, the level of label in cells declined to approximately 25% by 24 hours and returned to background levels by 48 hours. The membrane depolariser carbonyl cyanide m-chlorophenyl hydrazone (CCCP; (10um) accelerated this decline, such that at 6 hours BrdU signal was reduced by 70-100%. 24 or 48 hours of pretreatment with bafilomycin, a pharmacological inhibitor of early endosome acidification, partially reversed the CCCP stimulated loss of BrdU, and we confirmed by confocal microscopy that the BrdU in bafilomycin treated cells localized with LC3, a marker of early endosomes. Finally, we showed that treatment with urolithin, previously reported to inhibit mitophagy at high concentrations, showed a dose responsive inhibition of BrdU signal loss with a maximum of about 50% at 50uM. This assay represents a robust and simple method to evaluate agents accelerating mitophagy in postmitotic cells.

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