Acyl coa standards

Acyl-CoA species from the reaction were first separated from high molecular weight components using 3000 Da cutoff spin columns, then equilibrated with two volumes of 150 mM ammonium acetate/acetate buffer pH 4.5 containing 4% acetonitrile before injection into the high-performance liquid chromatography (HPLC). Acyl-CoA standards were purchased from Sigma-Aldrich then made into 10 mM stock in water. A Beckman System Gold HPLC system with an Agilent Pursuit XRs Ultra C18 column were used to separate the acyl-CoA species using a 150 mM ammonium acetate/acetate buffer pH 4.5 and a 4–24% acetonitrile non-linear gradient at 1 ml/min for 45 min. The gradients of acetonitrile were: 0–5 min 4%, 5–10 min linear increases from 4% to 16%, 10–35 min linear increases from 16% to 24%, 35–38 min remain at 24%, 38–40 min decrease from 24% to 4%, 40–45 min remain at 4%. In this method, a series of mixture of CoA and Acyl-CoA standards were run to calibrate the separations. The approximate retention times were: CoA, 13.5 min; acetyl-CoA, 15.5 min; malonyl-CoA, 8 min; succinyl-CoA, 15 min; glutaryl-CoA, 16 min, and pimeloyl-CoA, 18.5 min.

Primary human T cells were activated with Dynabeads and expanded for 7 days. T cells were maintained in culture at a concentration of 0.8–1.0 × 10⁶ cells/ml through regular counting. Where indicated, activated T cells (day3) were treated with 50μM ETO throughout expansion. The cells were harvested in 1 ml of ice-cold 10% w/v trichloroacetic acid solution, and every sample was spiked with an equal amount of ¹⁵N₁¹³C₃-labeled acyl-CoA internal standard (generated as described previously (Snyder et al., 2015)). Acyl CoA’s were extracted as described above. Absolute metabolite levels were determined by comparison with standard curves generated by serial dilution of acyl-CoA standards (Sigma-Aldrich) dissolved in trichloroacetic acid at known concentrations. Standards were spiked with internal standard and extracted in the same manner as the cell samples.