Oxygraph 2k oxygen electrode

High‐resolution respirometry was performed with an Oxygraph‐2 K oxygen electrode (Oroboros, Innsbruck, Austria). Basal respiration in intact cells was measured in supplemented DMEM (as described above), with non‐phosphorylating respiration (proton leak) measured in the presence of 5 mg·mL⁻¹ oligomycin and maximal respiration determined by the sequential addition of 1 μm aliquots of carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP). Non‐mitochondrial respiration was measured in the presence of 2 μm antimycin A. Spare respiratory capacity (maximal rate – basal rate) and cell respiratory control ratios (maximal/proton leak) were calculated according to [72 (link)] using datlab software (version 4.51, Oroboros Instruments) and expressed as pmol O₂·s⁻¹·mg⁻¹ of whole cell protein. Significant differences were determined using Student's two‐tailed t‐tests.
High‐resolution respirometry was performed in isolated mitochondria with an Oxygraph‐2 K oxygen electrode in respiration buffer (255 mm Mannitol, 75 mm Sucrose, 10 mm KCl, 10 mm Tris HCl pH 7.2, 5 mm KH₂PO₄ pH 7.2) with the addition of either 5 mm glutamate plus 5 mm malate, 5 mm pyruvate plus 5 mm malate, or 5 mm succinate, and State IV respiration recorded. State III respiration was recorded after the addition of 1 mm ADP.