The brain, heart, muscle, liver, testis, and MEF mitochondria were isolated in MS-EGTA buffer [225 mM mannitol, 75 mM sucrose, 5 mM Hepes, and 1 mM EGTA (pH 7.4); Sigma-Aldrich]. The various tissues were minced into 2 mm by 2 mm pieces and then homogenized using a glass and Teflon tissue homogenizer (8 to 15 strokes, depending on the tissue) on ice and in MS-EGTA buffer. Tissue homogenates were then subjected to a 2500g centrifugation for 5 min (1×), and the supernatants were then centrifuged at 11,500g for 10 min. The MEFs were grown to confluency on four Nunc Bioassay Dishes (Sigma-Aldrich) and then harvested and pelleted in MS-EGTA buffer. The cells were then suspended in 7 ml of buffer and homogenized with a glass and Teflon tissue homogenizer (10 strokes). MEF homogenates were then subjected to a 2500g centrifugation for 5 min (2×), and the supernatants were then centrifuged at 11,500g for 10 min. In both situations, pellets were then washed and centrifuged 2× (11,500g) and then snap-frozen or suspended in KCl buffer [125 mM KCl, 20 mM Hepes, 2 mM MgCl2, 2 mM KH2PO4, and 40 μM EGTA (pH 7.2); Sigma-Aldrich].

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