Mitochondria isolation kit for tissue

Mitochondria were isolated from BAT using the Mitochondria Isolation Kit for Tissue (Abcam) and applied for the following proteomics analysis. The following biological replicates were used for mitochondrial proteomics: n=3 for both young BAT and old BAT, n=4 for control BAT, and n=3 for Adipo-Bola3 BAT.

ETC activity was measured by using the protocol published previously ⁵⁴ . In brief, mitochondria were isolated from the BAT of mice using the Mitochondria Isolation Kit for Tissue (Abcam) and were resuspended in 200 μl of isolation buffer provided by the kit. After protein quantification by the BCA method, the mitochondrial suspension was diluted with the isolation buffer at concentration 0.2 mg/ml, seeded into a 24-well plate (10 μg/50 μl/well), and adhered to the bottom of the plate by centrifugation 2,000 x g at 4°C for 20 min. Immediately prior to the measurement, hypotonic mitochondrial assay buffer supplemented with 50 mM KCl, 4 mM KH₂PO₄, 5 mM MgCl₂, 5 mM HEPES, 1 mM EGTA, 10 mM Pyruvate, 5 mM Malate and 4% fatty-acid-free BSA was added to each well. For the measurement of complex activities of I and II, mitochondria were sequentially injected with 2 μM rotenone (complex I inhibitor) and 10 mM succinate (complex II substrate). For the measurement of complex III activity, hypotonic mitochondrial assay buffer with 10 mM Malonate (complex II inhibitor) was added to each well and mitochondria were sequentially treated with 2 μM rotenone, 10 mM succinate and 5 μM antimycin A.