Measuring Mitochondrial Respiration and Glycolysis

Using the Seahorse XF analyzer (Seahorse Bioscience, North Billerica, MA), in real time, mitochondrial oxidative phosphorylation and glycolytic flux can be analyzed by measuring OCR and ECAR of cells as they respond to substrates and metabolic inhibiting agents according to manufacturer’s instructions [34 (link)]. ATP synthase inhibitor oligomycin (a complex V blocker) is added to inhibit coupled respiration. FCCP (a mitochondrial uncoupler) is added to collapse mitochondrial membrane potential (Δψm). Rotenone (an inhibitor of complex I of the electron transport chain) and antimycin A (an inhibitor of complex III of the electron transport chain) were add to block mitochondrial respiration completely. To analyze real-time glycolytic rates, Seahorse XF glycolytic rate assay utilizes both (extracellular acidification rate) ECAR and OCR measurements to evaluate the glycolytic proton efflux rate (glycoPER) of the cells, in which the cells were incubated in glucose-free media followed by the addition of rotenone, antimycin A, and finally 2-deoxyglucose (2-DG, glycolysis inhibitor). OCR and ECAR were described as absolute rates (pmoles/min for OCR and mpH/min for ECAR) and normalized against cell counts as a percentage of the baseline oxygen consumption.