Seahorse xf dmem ph 7.4 medium

One day prior to the assay, CD8 T cells were cultured in substrate-limited growth medium (Seahorse XF DMEM [pH 7.4] medium [Agilent] supplemented with 0.5 mM glucose, 1 mM glutamine, 1% fetal bovine serum, 0.5 mM L-carnitine, and 5 U/mL human IL-2). On the day of the assay, cells were washed and resuspended in Seahorse XF DMEM medium supplemented with 2 mM glucose and 0.5 mM L-carnitine. Cells (10⁵ cells/well) were then plated onto Cell-Tak-coated Seahorse cell plates. The OCR was measured in response to Omy (1.5 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 1.5 μM) and rotenone/antimycin A (0.1 μM and 1 μM, respectively) (all from Sigma-Aldrich). Measurements were taken using a Seahorse XFp analyzer (Agilent).

Two hours before the assay, CD8 T cells were stimulated with 2 μg/mL anti-CD3 mAb (145-2C11) crosslinked with 1 μg/mL anti-hamster immunoglobulin G (IgG) (MP) Ab in complete medium. They were then harvested, washed, and resuspended in Seahorse XF DMEM (pH 7.4) medium (Agilent) supplemented with 10 mM glucose, 1 mM sodium pyruvate, and 2 mM glutamine. Cells (10⁵ cells/well) were plated onto Seahorse cell plates coated with Cell-Tak (Corning). The glycolytic rate test was performed by measuring the glycolytic proton efflux rate (GlycoPER), which correlates with lactate accumulation, in response to rotenone/antimycin A (0.5 μM each) and 2-DG (50 mM) (all from Sigma-Aldrich). Measurements were taken using a Seahorse XFp analyzer (Agilent).