Forty μg of mitochondria were loaded per well of a Seahorse XFp plate in 40 μl of mitochondrial assay buffer (MAS: 220 mM d-mannitol, 70 mM sucrose, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% w/v fatty acid-free BSA, pH 7.2)60 containing 10 mM malate, 10 mM glutamate, 4 mM ADP and 1,500 μM BHAM. The plate was centrifuged at 2,000g for 20 min at 4 °C. Then, 110 μl MAS containing malate, glutamate, ADP and BHAM was added to each well. Rotenone, succinate (10 mm), antimycin A or a combination of ascorbate (10 mM) and TMPD (100 μM) were injected as indicated.
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