Mitochondrial Respiration and Mitophagy Assay

Mitochondrial respiration was measured through analysis of mitochondrial oxygen consumption rate (OCR), as described previously by our group 36 . In brief, cells were seeded at 40 000 cells/well on 96- well XFe96 cell culture microplates and cultured for 48 hours under an XFe96 extracellular flux analyzer (Agilent Technologies). For respiration assays, cells were incubated in a CO₂- free environment for 1 hour, and OCR was measured every 3 minutes for the next 90 minutes. First, OCR was quantified in basal conditions (20 mmol/L glucose), then with 1 μmol/L oligomycin (ATP synthase inhibitor), next with 0.125 μmol/L FCCP (mitochondrial respiration uncoupler), and finally with 1 μmol/L rotenone/antimycin A (complex I and III inhibitors, respectively). Complex I Activity Assay Kit (ab109721) and Complex II Activity Assay Kit (ab109908), purchased from Abcam, were used to determine the activity of ETCs activity per manufacturer's protocol 40 (link).
Mt-Keima (pMT-mKeima-Red, #AM-V-251, MBL Medical & Biological Laboratories Co., ltd. Woburn, MA) is a ratiometric pH-sensitive fluorescent protein that is targeted into the mitochondrial matrix. Ratio (543/458nm) of mKeima emission light was calculated as an index of mitophagy.