Oxygen consumption rate and extracellular acidification rate

Mitochondrial function was analyzed by extracellular flux analysis using a Seahorse XF96 Analyzer (Agilent, Santa Clara, USA). CD4⁺ and CD8⁺ T cells were plated on a poly‐l‐lysine‐coated (Biochrom, # L7240, Berlin, Germany) 96‐well plates (#102601‐100, Agilent, Santa Clara, USA) in Assay Medium (containing Seahorse RPMI supplemented with 1 mM sodium pyruvate, 2 mM glutamine and 5.5 mM glucose for the Mito Stress Test or supplemented with 5 mM HEPES for the Glycolytic Rate Assay). All experiments were performed in triplicates using 200 000 cells per well. To measure extracellular acidification rate (ECAR), oxygen consumption rate (OCR) and glycolytic proton efflux rate (glycoPER) Mito Stress Test (#103015‐100) and Glycolytic Rate Assay (# 103344‐100) was performed. To analyze mitochondrial oxidative phosphorylation, final well concentrations of 1 µM Oligomycin, 0.75 µM FCCP and 0.5 µM Rotenone/Antimycin A were consecutively added through designated Seahorse cartridge compound delivery ports. For the evaluation of cellular glycolysis, 0.5 µM Rotenone/Antimycin A and 50 mM 2‐deoxy‐glucose were injected accordingly.