Seahorse oxygen consumption rate measurement.

The Seahorse XF cell mito stress test kit (Agilent, Santa Clara, CA) was used to measure the oxygen consumption rate (OCR) and to characterize mitochondrial respiration by extracellular flux analysis using an Agilent Seahorse XFe96 analyzer (Agilent). The Seahorse plate was coated with 0.01% poly-l-lysine, and 25,000 cells were allowed to adhere to each well from an overnight (16-h) culture in YNB (cell densities were tested and optimized prior to the assay). The cells were allowed to adhere for 30 min at 30°C before washing with fresh YNB. Additionally, 180 μl of Seahorse XF calibrant solution was added to each well of the Seahorse XF sensor cartridge to hydrate the XF utility plate. The hydrated cartridge was kept in a non-CO₂ incubator at 30°C for 24 h, thereby removing CO₂ from the media that would otherwise interfere with measurements. To allow the assay media to preequilibrate, 180 μl of YNB was added to each well, and the plate was placed in a 30°C in a non-CO₂ incubator 1 h prior to the assay. Mitochondrial respiration was analyzed by sequential injections of modulators (titration of each modulator was performed prior to the experiment): SHAM (5 mM) used to inhibit the alternative oxidase; oligomycin (10 μM) used to block ATP synthase; carbonyl-cyanide-4-(trifluoromethoxy)phenyhydrazone (FCCP) (4 μM) used to activate uncoupling of the inner mitochondrial membrane, allowing maximum electron flux through the electron transport chain; and a mix of rotenone (4 μM) and antimycin A (4 μM) used together to inhibit complexes I and III, respectively. These drugs were also used in the absence of SHAM; however, rotenone and antimycin A were added separately to characterize the impact of complex I and complex III inhibition individually. These modulators were diluted in YNB and loaded into the injection ports of the hydrated sensor cartridge corresponding to the order of injection 1 h prior to the assay.