Ms and ld columns

Spleens from 6–8-week-old mice were dissociated by mechanical dissociation using the plunger of a 2.5 mL syringe on a 70 μm cell strainer (Falcon). Red blood cells were lysed with ACK Lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA in H₂O, pH 7.4) for 2 min at RT. Following centrifugation cells were resuspended in RPMI 1640 (Gibco) supplemented with 1% heat-inactivated fetal calf serum (HI-FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomyocin and counted using a hemocytometer. Splenocytes were then washed and resuspended in 0.5% BSA and 2 mM EDTA in PBS at 1 × 10⁷ cells/mL for magnetic-activated cell sorting (MACS). CD4⁺ T cells were isolated by negative selection using a CD4⁺ T Cell Isolation Kit and LS columns (Miltenyi Bitoec), naive CD4⁺CD44⁻CD62L⁺ T cells were isolated using a Naive CD4⁺ T Cell Isolation Kit and LS columns (Miltenyi Biotec), and CD4⁺CD25⁻ conventional T cells (T_conv) and CD4⁺CD25⁺ regulatory T cells (T_reg) were isolated by sequential negative and positive selection using a CD4⁺CD25⁺ Regulatory T Cell Isolation Kit and LD and MS columns (Miltenyi Biotec) according to manufacturer’s protocols.