Mitochondrial Isolation from Cell Lines

We isolated mitochondria using a previously described method with some modifications [12 (link)]. In brief, L6 cells (ATCC; CRL-1458, Manassas, VA, USA) were homogenized using a 26 G syringe in SHE buffer (0.25 M sucrose, 20 mM HEPES [pH 7.4], 2 mM EGTA, 10 mM KCl, 1.5 mM MgCl₂ and 0.1% defatted bovine serum albumin [BSA] with 1 × protease inhibitor) and centrifuged at 1500×g for 5 min at 4 °C. The supernatant was centrifuged at 20,000×g for 10 min at 4 °C to obtain mitochondria. To isolate mitochondria from umbilical cord mesenchymal stem cells (UC-MSCs; IRB No. 201806-BR-029-03), UC-MSCs were homogenized using a 26 G syringe in SHE buffer and centrifuged at 1100×g for 3 min at 4 °C. The supernatant was centrifuged at 12,000×g for 15 min at 4 °C. The pellet was resuspended using SHE buffer without BSA, and the suspension was centrifuged at 20,000×g for 10 min at 4 °C to obtain mitochondria. For all experiments, the isolated mitochondria were stored at 4 °C until use.