PBMC Isolation, Fatty Acid Stimulation, and Antioxidant Treatment

Fifty milliliters of peripheral blood were collected into acid/citrate/dextrose containing tubes by venous puncture. PBMCs were purified by histopaque 1077 then frozen at −80 °C under controlled cooling conditions in a Mr. Frosty apparatus (Nalgene, Sigma Aldrich, St Louis, MO, USA). For multi-week storage, cells were moved to −170 °C following 1–7 days at −80 °C. PBMCs from the subjects were stimulated in vitro for 40 h with T cell-targeted αCD3/αCD28 Dynabeads (Thermo Fisher Scientific, 11132D, Waltham, MA, USA) at 2 μL Dynabeads per 100k cells. In some cultures, cells were co-treated with 400 µM palmitate (pal) (C16:0) coupled to fatty acid-free Bovine Serum Albumin (BSA) at a ratio of 2 mol palmitate to 1 mole BSA, or 400 µM oleate, or a combination of palmitate and oleate. These fatty acid concentrations mimic concentrations achievable in serum [13 (link)]. Control cells were treated with 1% BSA. The mitochondrial ROS scavenger MitoTempo (mito)(10 μM) or a general ROS scavenger N-acetyl cysteine (NAC) was added for the last 20 h of incubation (20 h post-stimulation) for some cultures. All treatments were done in RPMI media with 5 mM glucose (normoglycemic). Supernatants were collected and stored at −80 °C. Cells were assayed as outlined below.