2.3.3. General Conditions

The solubility of oxygen was assumed to be 223 μmol O2/L at 30 ◦C according to the Strathkelvin instruments manual. Respiration rates were expressed as nmol/min/mg protein. No correction of the natural drift of the electrode was made since a drift of less than 0.5% over 12 h is neglectable in our experimental setup.

#### 2.3.4. Quality Control for the Preparation Procedure

At the beginning of every experiment mitochondria were checked for leakage by the addition of 2.5 μM cytochrome c (Sigma-Aldrich Corporation, St. Louis, MO, USA) and 0.05 μg/mL oligomycin (Calbiochem by Merck KGaA, Darmstadt, Germany) at the beginning of every experiment. There was no increase in flux after the addition of cytochrome c, indicating integrity of the mitochondrial outer membrane. When ATP synthesis was inhibited by oligomycin, the mitochondria were transferred to state 2, which reflects the respiration rate compensating the proton leak. The lack of difference in O2 consumption after adding oligomycin compared to state 2 indicates that the inner membrane was intact and mitochondria were not damaged through the preparation procedure.
