Cellular oxygen consumption and extracellular acidification measurement

Cellular OCR and ECAR were determined with the XF Cell Mito Stress Test™ and XF Glycolysis Stress Test™, respectively, using an XFp Extracellular Flux Analyzer™ (Seahorse Bioscience, USA) [17]. Cells (2 × 10⁴ cells/well) were seeded into an XFp cell culture microplate, and OCR was assessed in glucose-containing XF base medium according to the manufacturer’s instructions. The sensor cartridge for the XFp analyzer was hydrated in a 37°C non-CO₂ incubator on the day before the experiment. Each extracellular flux assay included two control wells of medium alone (no cells) to provide the blanks for background correction. They are used to normalize the data to the background signal derived from non-cell-mediated process. For the OCR assay, injection port A on the sensor cartridge was loaded with oligomycin (complex V inhibitor, final concentration 1 μM), port B was loaded with carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, final concentration 2 μM), and port C was loaded with rotenone/antimycin A (inhibitors of complex I and complex III, final concentration 0.5 μM each). During sensor calibration, cells were incubated in a 37°C non-CO₂ incubator in 180 μL of assay medium (XF base medium with 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine, pH 7.4). The plate was immediately placed into the calibrated XFp extracellular flux analyzer for the Mito Stress test. The assay parameters were calculated as follows: OCR (basal respiration) = (last rate measurement before oligomycin injection) − (minimum rate measurement after rotenone/antimycin-A injection); OCR (maximal respiration) = (maximum rate measurement after FCCP injection) − (minimum rate measurement after rotenone/antimycin A injection); OCR (non-mitochondrial respiration) = (minimum rate measurement after rotenone/antimycin A injection); proton leak = (minimum rate measurement after oligomycin injection)–(non-mitochondrial respiration). For the ECAR assay, injection port A on the sensor cartridge was loaded with 10 mM glucose. During the sensor calibration, cells were incubated in a 37°C non-CO₂ incubator in 180 μL of assay medium (XF base medium with 2 mM L-glutamine, pH 7.4). The plate was immediately placed into the calibrated XFp extracellular flux analyzer™ for the Glycolysis Stress test. Oligomycin (1 μM) and 50 mM 2-deoxy-D-glucose were loaded for the measurement. ECAR was calculated as follows: ECAR (glycolysis) = (maximum rate measurement after glucose injection) − (last rate measurement before glucose injection).