Mitochondrial Electron Flow Analysis

Mitochondria were isolated from the soleus muscle and heart, and prepared based on the previously described procedure [36 (link),39 (link)]. Analysis was performed by Seahorse Metabolic Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA). For electron flow experiments, isolated mitochondria were diluted in cold MAS buffer (enriched with 10 mM pyruvate (Sigma-Aldrich, St. Louis, MO, USA)2 mM malate (Sigma-Aldrich, St. Louis, MO, USA), and 4 µM FCCP (Sigma-Aldrich, St. Louis, MO, USA). A mitochondrial suspension of 25 µL was placed into Seahorse plate wells and centrifuged at 2000× g for 15 min at 4 °C. The concentration of mitochondrial protein was 6 µg per well. After centrifugation, 180 µL of prewarmed MAS buffer supplemented with pyruvate, malate, and FCCP was added to each well, and the plate was then placed into a non-CO₂ incubator for 8 min. The Seahorse cartridge was filled with the following reagents: 2 µM Rotenone (Sigma-Aldrich, St. Louis, MO, USA), 2 mM succinate (Sigma-Aldrich, St. Louis, MO, USA), 4 µM Antimycin (Sigma-Aldrich, St. Louis, MO, USA), and a mix of 10 mM ascorbate (Sigma-Aldrich, St. Louis, MO, USA) and 100 µM TMPD (Sigma-Aldrich, St. Louis, MO, USA).