Mitochondrial dysfunction results from oxidative stress in skeletal muscle of diet-induced insulin resistant mice

Mitochondria isolation

Muscle was thawed in isolation buffer (Mannitol 210mM, Saccharose 70mM, Tris 50mM, EDTA 10mM and BSA 0.5%, pH=7.4) and cut in small pieces. Then, it was digested 15 minutes with trypsin, under agitation, and washed 2 times with the isolation buffer. After each wash, the tissue was centrifuged two minutes, at 70g. The tissue was homogenised with a conical glass grinder (VWR International) in 1ml of isolation buffer. The homogenate was centrifuged 10 minutes at 820g. Then the supernatant was centrifuged 20 minutes at 6800g. The pellet was resuspended in 1ml of suspension buffer (Mannitol 225mM, Saccharose 75mM, Tris 10mM, and EDTA 0.1mM, pH=7.4) and centrifuged 10 minutes at 820g. The mitochondria were then pelleted by centrifuging the supernatant 20min at 6800g, and resuspended in 50μl of the same buffer. Western-blotting analysis was used to detect cytochrome C (Santa Cruz, 1/100) content in cytosolic and mitochondrial fractions.