Easysep mouse cd4 t cell negative selection and isolation kit

FACS-sorted LECs from Sp19F-infected mice were co-cultured with OT-II CD4⁺ T cells (1:10 ratio) with 25μg/mL of OVA_323-339 peptide (Anaspec) in media containing 25% DMEM (Gibco), 25% F-12 (Gibco), 50% RPMI 1640 (Gibco) supplemented with 10% Heat inactivated FBS, Penicillin/Streptomycin (100 U/mL, Gibco), 1X Non-essential amino acids (Gibco), 2 mM L-Glutamine (Glutamax, Gibco), 1 mM Na-pyruvate (Gibco), 10 mM HEPES (Gibco), 0.2 μM beta-mercaptoethanol, 10 μg/mL Insulin, 50 mM Hydrocortisone, 100 μg/mL Transferrin, 1 mM Na-selenite, and 1 mM beta-estradiol for 8 h at 37 °C 5% CO₂ incubator. For assays with C57BL/6J LECs, EasySep™ Mouse CD4⁺ T Cell negative selection and Isolation Kit (StemCell Technologies, Cat# 19852) was used to enrich OT-II CD4⁺ T cells from spleens of OT-II TCR transgenic mice and 10 μg/mL of antimouse MHC-II antibody (Biolegend) was used for MHC-II blockade. For assays with MHC-II^ΔEpi LECs, EasySep™ Mouse CD4⁺ T Cell negative selection and Isolation Kit (StemCell Technologies, Cat# 19852) was used to enrich OT-II CD4⁺ T cells from spleens of OT-II TCR transgenic mice. This was followed by MHC-II blockade of the enriched CD4⁺ T cell suspension to block any excess MHC-II expressing splenocytes before rigorous washing and coculture with LECs for 8 h.