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WAT mitochondria isolation To isolate mitochondria, fresh gWAT was minced then homogenized using a drill-operated Teflon pestle in ice-cold MSHE buffer (70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EDTA, pH 7.2) containing 0.5% FA-free BSA

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Once thawed: Centrifuge briefly (30 seconds at 10,000 rpm) to collect any condensation from the tube walls Prepare fresh dilutions in the desired experimental buffer (PBS, cell culture media, etc.) Maintain on ice during experiment setup Use within 24 hours of dilution