Metabolic measurements

Real-time measurement of pH and oxygen concentration for the determination of ECAR and OCR were performed in XF96 plates with the XF Extracellular Flux Analyzer (Seahorse Bioscience-Agilent (Santa Clara, CA). Key parameters of mitochondrial function were measured by using specific mitochondria respiration inhibitors (Seahorse XF Cell Mito Stress Test Kit, 103015–100, Agilent Technologies) while recording OCR values. The kit contains: oligomycin (ATP synthase/complex V inhibitor), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (uncoupling agent responsible for disrupting of the mitochondrial membrane potential) and rotenone (complex I inhibitor). Contribution of glycolysis to ECAR was determined by adding 2-DG (glycolysis inhibitor; Sigma Aldrich, D8375). The results obtained were normalized by the number of cells.