Measurement of Mitochondrial Oxygen Consumption

Mitochondrial respiration was measured polarographically at 28°C with a computer-controlled Clark-type O₂ electrode (Hansatech Instruments Ltd, Norfolk, UK) in 0.5 mL respiratory buffer containing (in mM): KCl 130, K₂PH₄ 5, MOPS 20, EGTA 2.5, Na₄P₂O7 0.001 EDTA, and 0.1% BSA (pH 7.2 adjusted with potassium hydroxide). Mitochondrial suspension (0.5 mg/mL) were added in respiratory buffer in the presence of complex I substrate glutamate (5 mM) plus malate (5 mM).