Xf base medium minimal dulbecco s modified eagle s medium

Measurements of the oxygen consumption and extracellular acidification rates were performed with a Seahorse XFp or XFe96 Extracellular Flux Analyzer (Agilent Technologies), as described previously.²⁶ (link) CD4⁺ and CD8⁺ T cells were isolated from fresh blood of healthy donors and stimulated with α-CD3 and α-CD28 (both 4 μg/ml plate-bound) for indicated time points. Activated T cells were cultured in XF Base Medium Minimal Dulbecco’s Modified Eagle’s Medium (Agilent Technologies) containing 10 mM glucose, 2 mM l-glutamine and 1 mM sodium pyruvate (all Merck). The oxygen consumption and extracellular acidification rates were simultaneously acquired under basal conditions and in response to 1 µM oligomycin, 1 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), 100 nM rotenone plus 1 µM antimycin A and 50 mM 2-desoxy-glucose (all Merck). When indicated, CD4⁺ and CD8⁺ T cells were isolated from frozen PBMCs of healthy donors and patients (Supplementary Tables 1–6). Before the oxygen consumption and extracellular acidification rates were simultaneously assessed using the XF96 Extracellular Flux Analyzer, T cells were either left unstimulated or were short-term stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA, Merck) and 500 ng/ml ionomycin (Cayman Chemical) for 2.5 h. Data analysis was performed with Wave Software (Agilent Technologies).