Mitochondrial respiration assay

We used an XF Mitochondrial Stress test kit (Seahorse Bioscience Inc.) with some modifications to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). B16F10 murine melanoma cells were seeded into the lower 96-well plate of the XFe96 (Seahorse Bioscience) extracellular flux assay kit. The optimal seeding density was determined prior to the assay to be around 20,000 cells/well. The cells were washed, and the medium was replaced with glucose- and glutamine-free medium. The pre-treatment was started 1 h prior to the assay. The plate was incubated at 37 °C in an oxygen-rich atmosphere, while the upper plate was calibrated according to the kit protocol. The baseline oxygen level and pH of the extracellular space were measured three times in 20 min prior to the injection of glucose and glutamine (both from Sigma-Aldrich). Final concentrations of the standards were 25 mM glucose and 5 mM glutamine, while the concentrations of the modulators of mitochondrial function were 3.0 µM oligomycin, 0.25 µM FCCP, and 10 µM antimycin-A and rotenone.