Aerobic Respiration and Glycolysis Measurement

Real-time measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored using Vero cells infected with RVPs, with the XFe extracellular flux analyzer (Agilent Seahorse Technologies, Santa Clara, CA, USA). Briefly, 10,000 Vero cells/well were plated into XF96 microplates for ECAR measurement, and 15,000 cells/well for OCR measurement. Prior to the analysis, media was changed to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 10 mM glucose (Agilent Seahorse Bioscience), 1 mM sodium pyruvate and 2 mM L-glutamine for OCR (readout of mitochondrial respiration) and to XF DMEM (Agilent Seahorse Technologies, Santa Clara, CA, USA) supplemented with 2 mM of glutamine for ECAR monitorization as an indirect readout of acidification by the production of lactate (aerobic glycolysis). For ECAR measurement the following sequential injections were performed: glucose (10 mM), oligomycin (2 µM), and 2-DG (75 mM). For OCR, sequential injection of oligomycin (2 µM), trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP; 1.2 µM), and rotenone/antimycin A (0.5 µM) as previously described [21 (link), 22 (link)].