Seahorse xf base medium

Cellular oxygen consumption (OCR) and extracellular acidification rates (ECAR) were determined in Seahorse XF Base Medium supplemented with 25 mM glucose (Sigma-Aldrich), 2 mM L-glutamine and 1 mM sodium pyruvate (both from BioWest) using the XF cell Mito Stress Kit (SeaHorse Bioscience), in an XF24 Extracellular Flux Analyzer (SeaHorse Bioscience; Agilent Technologies). Fatty acid oxidation (FAO) was determined in Krebs-Henseleit buffer (KHB) supplemented with 0.5 mM carnitine (Sigma-Aldrich) and 2.5 mM glucose, using palmitate as substrate, in the Agilent Seahorse XF96 Extracellular Flux Analyzer.
Lactate levels were determined enzymatically in extracts from T_CTRL, T_ACT and T_{ACT + PD1} cells after 48 h stimulation, using a fluorometric lactate assay kit (Cell Biolabs) according to supplier’s protocol; fluorescence was quantified in a Filter Max F5 microplate reader (Molecular Devices) at 530/590 nm excitation/emission. A lactate standard curve was generated in all assays and used to extrapolate relative fluorescent units (RFU) measured in the samples.

Oxygen consumption rate and ECAR were measured using Seahorse XF24 or XF96 analyzers in purified CD8 T cell subsets (4 × 10⁵ or 2.5 × 10⁵ purified cells, respectively) that were allowed to rest overnight after FACS-sorting in TexMACS buffer at 37°C/5% CO₂. The assay was performed in Seahorse XF-base medium supplemented with 10 mM glucose (Sigma), 2 mM glutamine (Life Technologies), and 1 mM pyruvate (Life Technologies). Mitochondrial stress assay was performed by adding successively oligomycin (1.5 µM; Sigma), CCCP (1 µM; Sigma), and Antimycin A + Rotenone (1 µM each; Sigma). To assess OCR and ECAR upon polyclonal stimulation, PMA (50 ng/mL; Sigma) and Ionomycin (500 ng/mL; Sigma) were added 75′ after the start of the experiment. 2-DG (250 mM; Sigma) and oligomycin (1.5 µM; Sigma) were added before the stimulation with PMA-Iono.

LCLs were stained with CD54 (ICAM-1) antibody (PE, Biolegend #HA58) according to the supplier’s manual. Then, cells were sorted on a Beckman Coulter Astrios cell sorter by anti-CD54 fluorescence, with ICAM-1-high and ICAM-1-low being defined as the top 15% and bottom 15%, respectively. 24 hr after sorting of ICAM-1-high and ICAM-1-low LCLs, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using the Seahorse XF24 extracellular flux analyzer (Agilent Technologies) Cell Energy Phenotype Test. Suspension LCLs were attached to culture plates by using Cell-Tak (BD Bioscience). ECAR and OCR were measured in Seahorse XF Base Medium supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose (Sigma Aldrich). ECAR and OCR values were normalized to cell number. For stress measurements, ECAR and OCR were measured over time after injection of oligomycin and FCCP. Metabolic potential measures the ability of cells to meet energetic demands under conditions of stress and is the percentage increase of stressed over baseline ECAR or OCR.