Isolation of mitochondria

Heart mitochondria were isolated from 2.5–3 month old mice as described with slight modifications [34]. The isolation process was carried out at 4°C. Briefly, hearts were collected in ice-cold PBS supplemented with 10 mM EDTA and minced. Following incubation with 0.05% trypsin for 30 minutes, samples were centrifugated at 200 × g for 5 min. This was followed by homogenization in buffer containing 67 mM sucrose, 50 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, 0.2% BSA, pH 7.4 and the homogenate was centrifuged at 700 × g for 15 min. The supernatant was again centrifuged at 7,000 × g for 15 min and the pellet resuspended in 250 mM sucrose, 3 mM EGTA and 10 mM Tris-HCl, pH 7.4. The solution containing the mitochondria was centrifuged at 7,000 × g for 15 min and the pellet containing mitochondria was resuspended in minimum amount of buffer. A Bradford assay was performed to determine the mitochondria concentration [34]. To determine if the mitochondria were functional, enzymatic activity assays (complex I, II or III) were carried out immediately with the respective inhibitors. Flash frozen mitochondria were also used for this study; the frozen mitochondria showed similar enzymatic activity when compared to activity of freshly isolated mitochondria [35].