Naive CD4+ T Cell Isolation and Lipid Uptake

Naive (CD44^loCD62L^hi) CD4⁺ T cells were purified from the spleens of mice. After lysis of red blood cells, the CD4⁺ T cells were obtained using a CD4⁺ T cell isolation kit with anti-CD4 microbeads (Miltenyi Biotec), and naive CD44^loCD62L^hi cells were then further sorted to more than 99.5% purity using a FACS Aria cell sorter (BD Biosciences). Naive CD4 T cells were plated onto 24-well tissue culture plates (Costar) pre-coated with 10 μg ml⁻¹ agonistic anti-TCRβ antibody (H57-597) with 1 μg ml⁻¹ agonistic anti-CD28 antibody (clone 37.51, Biolegend). Non-polarized Th cell cultures contained IL-2 (15 ng ml⁻¹), anti-IL-4 antibody (BD Biosciences, BVD4-1D11) (1 μg ml⁻¹) and anti-IFNγ antibody (Biolegend, R4-6A2) (1 μg ml⁻¹). Oleic acid (Sigma-Aldrich) was dissolved in DMSO to a final concentration of 100 mM and was complexed to BSA. For lipid uptake experiments, naive CD4 T cells were cultured under indicated conditions in the presence of Bodipy FLC16, Bodipy LDL, or Bodipy 493/503, and mean fluorescence of Bodipy was measured by flow cytometry31 (link)32 (link)33 (link).