Lipid supplement assay

LPC (16:0 LPC, 16:0 αLPC, and 16:0 pLPC), PE (16:0 lyso PE), and PS (16:0 lyso PS) were purchased from the Avanti Polar Lipids Inc. C2-Cer was purchased from Sigma-Aldrich. All lipids were dissolved in chloroform, evaporated under a gentle stream of nitrogen, and then immediately dissolved in an ethanol vehicle (0.1%). Naïve CD4⁺ T cells were first treated with or without the lipids (5 or 10 μM) for 1 hour and then cocultured with T_reg cells at a ratio of 4:1 in anti-CD3–coated (2 μg/ml) plates for 3 days. The treated naïve CD4⁺ T cells were then stained for SA-β-gal and Oil Red O.