Mitochondrial Respiration Protocols for Cellular Analysis

Cells and tissue were prepared for mitochondrial respiration as described previously [9 (link)] before being transferred to respirometer chambers using the Oroboros O2K oxygraph (Oroboros, Innsbruck, Austria). Electron flow through complex I was supported by glutamate + malate (10 mM and 2 mM, respectively) to determine leak oxygen consumption (GML). Following stabilization, adenosine diphosphate (ADP) (2.5 mM) was added to determine oxidative phosphorylation capacity (GMD). Succinate was added (GMSD) for complex I + II electron flow into the Q-junction. Lastly, residual oxygen consumption was measured by adding antimycin A (2.5 μM) to block complex III action, effectively stopping any electron flow, which provides a baseline rate of respiration. Respiratory control ratio (RCR) was determined as the ratio of GM_P/GM_L. Following respiration protocol, samples were removed from the chambers and used for further analysis, including protein quantification.