Measurement of mitochondrial respiration

OCR was measured with a Seahorse XF^e96 Extracellular Flux Analyzer (Agilent, Santa Clara, CA). Cells were seeded in 96-well Seahorse tissue culture microplates in growth media at a density of 80 000 cells per well. To ensure equal cell numbers, cells were seeded in cell culture plates pre-coated with Cell-Tak (BD Biosciences, San Jose, CA). All cultured fibroblasts were measured with four to six wells per cell. Then, the entire experiment was repeated. Before the Seahorse assay, cells were incubated for 1 h without CO₂ in unbuffered DMEM. Initial OCR was measured to establish a baseline at the resting state (basal respiration) followed by injection of oligomycin (an inhibitor of ATP synthase) that reduces OCR, representing ATP turnover. Subsequent injection of 300 nM carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, Seahorse XF Cell Mito Stress Test Kit; Agilent, Santa Clara, CA) dissipates the proton gradient and allows maximum respiration. The rise in OCR upon FCCP addition represents mitochondrial reserve capacity. Finally, rotenone and antimycin A were added to effectively disable the ETC and inhibiting the total mitochondrial respiration. The remaining OCR represents non-mitochondrial respiration. The difference between oligomycin- and rotenone and antimycin A-responsive OCR reflects proton leak (see Fig. 1A for more details). Data are reported in pmol of O₂ reduced/min.