Seahorse XF96 analysis of the extracellular acidification rate (ECAR)

A Seahorse XF96 analyzer (Agilent, Santa Clara, USA) was used to monitor the ECAR continuously. Two days prior to performing the experiment, the cells were grown in a XF96 well plate at a density of 2 × 10³ cells/well at 37 °C in 5% CO₂. One day prior to the experiment, 200 µl of XF calibration buffer was added to each well of the XF cartridge, followed by incubation in an atmosphere of 0% CO₂ at 37 °C overnight. Thirty minutes prior to the experiment, the cells were washed with PBS and 200 µl of XF assay medium was added to each well, followed by incubation for 30 min in an atmosphere of 0% CO₂ at 37 °C. For the XF glycolysis stress analysis, 300 mg/L glutamine was added to the XF assay medium. After equilibration time, different compounds (10× final concentration) were added to the injection ports. The substrate consisted of 2 µM oligomycin, 10 mM glucose, and 50 mM 2-deoxyglucose. All the reagents for ECAR were purchased from Seahorse Bioscience.