Oxygen consumption measurement

Mitochondria respiratory rates were measured into the O₂K oxygraph chambers (Oroboros, Instruments Oroboros, Innsbruck, Austria) at 37°C in the respiration medium MiR06 [0.5 mM EGTA, 3 mM MgCl₂, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH₂PO₄, 20 mM Hepes, 110 mM sucrose, fatty acid–free bovine serum albumin (1 g/liter), and catalase (280 U/ml) (pH 7.1)]. To detect the electron flow through respiratory chain complexes, titrations of all of substrates, uncouplers, and inhibitors were added in series as previously reported (63, 64).

Briefly, we added pyruvate (10 mM) and malate (2 mM) to obtain state 2 respiration. Then, we injected ADP (2.5 mM) to evaluate state 3 respiration, and the further addition of succinate (10 mM) allowed the measurement of complex I (CI)– and CII-driven respiration (state3 + Succ). Then, CI was inhibited by injecting rotenone (0.5 μM) (state3 + Succ+ Rot) and state 4 was measured by administration of oligomycin (2 μg/ml). Last, we added antimycin A (2.5 μM) to inhibit CIII obtaining a residual oxygen consumption. Cytochrome c (10 μM) was added to test the integrity of the outer mitochondrial membrane. Oxygen fluxes were corrected by subtracting residual oxygen consumption from each steady state.

For experiments with cell culture, 1 × 10⁶ viable cells were transferred into oxygraph chambers. When oxygen consumption reached a plateau, a steady-state level was obtained displaying routine respiration. The addition of oligomycin (0.5 μM) resulted in leak respiration. Subsequently, the proton gradient was released by stepwise titration (0.5 μM each step) of the uncoupler carbonyl cyanide-4-(trifluoromethoxyphenylhydrazone) until the maximum respiration was achieved. The addition of 0.5 μM rotenone and 2.5 μM antimycin A blocked mitochondrial respiration, showing residual oxygen consumption. Initial addition of pyruvate (10 mM) and malate (2 mM) was performed to test the integrity of the plasma membrane. Oxygen fluxes were corrected by subtracting residual oxygen consumption from each steady state.