Isolation of mitochondria from mouse tissue

Mitochondria were prepared from mouse hearts as previously described (5). Briefly, tissue was minced and resuspended in MSH buffer [210 mM mannitol, 70 mM sucrose, 5 mM Hepes (pH 7.5)] supplemented with 1 mM EDTA. Homogenization was performed with a glass/glass dounce homogenizer and centrifuged at 600g for 10 min at 4°C. The supernatant was removed and respun at 600g for 10 min. The resulting supernatant was removed and centrifuged again at 5500g for 20 min at 4°C. The mitochondrial pellet was washed by resuspension in fresh MSH buffer without EDTA and centrifuged again at 5500g for 20 min. The mitochondrial pellet was referred to as the mitochondrial fraction.