T-cell Proliferation Modulation by Fatty Acids

PBMCs were labeled with Cell Proliferation Dye (CPD) eFluor450 (Thermo Fisher Scientific) and stimulated for 4 days with plate-bound anti-CD3 (clone OKT3; Thermo Fisher Scientific). In some experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) supplemented with BSA (10% v/v; Sigma-Aldrich) for 1 day in the absence or presence of palmitic acid (PA; 300 μM; Sigma-Aldrich), and in other experiments, cells were precultured in AIM-V medium (Thermo Fisher Scientific) without BSA supplementation for 2 days in the absence or presence of rosiglitazone (40 μM; Sigma-Aldrich). Proliferation was measured using flow cytometry to quantify the dilution of CPD. Sample size calculation, based on available data for T-cell proliferation (5 (link)), suggested a size of 10 individuals per group to detect a 50% difference in CPD low (i.e. proliferating) cells between middle-aged and old individuals with a power of 80% using a one-sided significance level of 5%. To assess the effect of fatty acids on resting cells, PBMCs were cultured up to 48 hr in AIM-V medium supplemented with 10% BSA in the absence or in the presence of palmitic acid (300 or 3000 μM).