4.5. Mitochondria Oxygen Consumption Rate

Mitochondria oxygen consumption rate (OCR) was measured with the XF24 Extracellular Flux Analyzer (Seahorse Bioscience, Agilent, Santa Clara, CA, USA), according to the standard protocol. Briefly, at least 3 months after each knockout, cells were seeded one day prior to the assay in a 24-well XF plates at a density of 2 × 10⁵ cell/well and incubated overnight at 37 °C, 5% CO₂. Twenty-four hours later, cells were incubated with Seahorse XF Base medium supplemented with 10 mM glucose, 2 mM L-glutamine and 1mM sodium pyruvate at pH 7.4 and calibrated for 1 h at 37 °C in the absence of CO₂. Hydration of the sensor cartridge was performed one day prior to the assay at 37 °C in the absence of CO₂. OCR was evaluated in a time course set-up where the following compounds were sequentially injected in the following order: oligomycin (1 µM final concentration), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.5 µM final concentration), and rotenone plus antimycin A (0.5 µM final concentration). Rates were normalized to protein concentration measured according to the Bradford method (Bio-Rad, Hercules, CA, USA). Three to five wells from each cell line were measure in a total of n = 3 experimental assays. Values for each parameter were calculated as the difference of OCR measures after and before injection:

Non-mitochondrial respiration was calculated as the average of OCR measurements after rotenone and antimycin A injection;

Basal respiration is calculated as the difference between non-mitochondrial respiration and the third point of baseline cellular oxygen consumption;

Maximal respiration corresponds to the difference between the average OCR value after FCCP injection and the non-mitochondria respiration;

Spare capacity rate (SCR) is the difference between maximal and basal respiration values.