Seahorse xf rmpi 1640 medium

Extracellular flux analysis of T cell activation was performed by adopting previously outlined protocols⁶⁶ . In short, in vitro activated T cells were spun onto a Cell-Tak (Thermo Fisher) coated XF96 cell culture microplate (Agilent) with a density of 100,000 or 150,000 cells/well and rested in Seahorse XF RMPI 1640 medium supplemented with 2 mM L-glutamine, 2 mM sodium pyruvate and 25 mM glucose (all Agilent) for 1 h in a non-CO2 incubator at 37 ºC. ECAR and OCR were measured using a Seahorse XF96 extracellular flux analyzer (Agilent). Oligomycin (1 μM), fluoro-carbonyl cyanide phenylhydrazone (FCCP; 1.5 μM) and rotenone (0.5 μM) together with antimycin A (0.5 μM) were sequentially injected to establish baseline parameters. Extracellular flux analysis of macrophages was performed as outlined previously⁶⁷ , including an additional injection of 25 mM glucose. Raw data was imported into the R environment in order to calculate basal glycolysis respiration rates as described⁶⁸ . Data was normalized by cell number. For linear regression between extracellular flux analysis values and mass cytometry values, both were asinh transformed with a cofactor of 5.