Isolation of Mouse Liver Mitochondria

Ethics Statement: Animal housing, euthanasia, and tissue harvest procedures were conducted in accordance with and approved by the UCSD Institutional Animal Care and Use Committee (protocol #S09186) and the Buck Institute Animal Care Committee (protocol #10180). Mitochondria from C57bl/6 (male and female) mice aged 4–6 weeks were isolated by two similar differential centrifugation methods, based upon Schnaitman and Greenawalt [14] (link) or Chappell and Hansford [15] . Specifically, the liver was extracted and minced in ∼10 volumes of MSHE+BSA (4°C), and all subsequent steps of the preparation were performed on ice. The material was rinsed several times to remove blood. The tissue was disrupted using a drill-driven Teflon glass homogenizer with 2–3 strokes. Homogenate was centrifuged at 800 g for 10 min at 4°C. Following centrifugation, fat/lipid was carefully aspirated, and the remaining supernatant was decanted through 2 layers of cheesecloth to a separate tube and centrifuged at 8000 g for 10 min at 4°C. After removal of the light mitochondrial layer, the pellet was resuspended in MSHE+BSA, and the centrifugation was repeated. The final pellet was resuspended in a minimal volume of MSHE+BSA. Total protein (mg/ml) was determined using Bradford Assay reagent (Bio-Rad). Typically, ∼7.5 mg of mitochondria (100 µl volume) was obtained from a single mouse liver. In separate studies in which respiratory rates in the Seahorse and the Rank Clark electrode system were compared, mouse liver mitochondria were isolated according to Chappell and Hansford [15] in 250 mM Sucrose, 5 mM Tris and 2 mM EGTA (STE) on ice. Tissue was homogenized 10 times with a Teflon-glass homogenizer, and the homogenate was centrifuged at 1000 g for 3 minutes (4°C). The supernatant was collected and centrifuged at 11,600 g for 10 minutes. The pellet was resuspended in STE after discarding the whitish layer. The above step was repeated two times to get the final mitochondrial pellet. 8–10 mg of mitochondrial protein was obtained from each mouse liver and resuspended in 400–500 µl of STE.