Seahorse XFe96 Metabolic Flux Analysis

Real-time oxygen consumption rates (OCRs) and extracellular acidification rates (ECAR) rates were determined using the Seahorse Extracellular Flux (XFe96) analyzer (Seahorse Bioscience, USA) (19–21). Briefly, 2 × 10⁴ cells per well were seeded into XFe96 well cell culture plates after sorting, and incubated for 12 h to allow cell attachment. After 12 h of incubation, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10 mM glucose, 1 mM Pyruvate, 2 mM L-glutamine, and adjusted at 7.4 pH). Cells were then maintained in 175 μL/well of XF assay media at 37°C, in a non-CO₂ incubator for 1 h. During the incubation time, we loaded 25 μL of 80 mM glucose, 9 μM oligomycin, and 1M 2-deoxyglucose (for ECAR measurement) or 10 μM oligomycin, 9 μM FCCP, 10 μM rotenone, 10 μM antimycin A (for OCR measurement), in XF assay media into the injection ports in the XFe96 sensor cartridge (20, 21). Measurements were normalized by protein content (SRB assay) and Hoechst 33342 content. Data sets were analyzed using XFe96 software and GraphPad Prism software, using one-way ANOVA and Student's t-test calculations. All experiments were performed in quintuplicate, three times independently.