4.4. Cell Culture and Cell Enrichment by Magnetically Activated Cell Sorting (MACS)

A single-cell suspension of splenocytes and MLN cells was prepared in RPMI complete medium consisting of RPMI 1640 (Gibco BRL, Gaithersburg, MD, USA) medium supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 100 units/mL penicillin-streptomycin, and 5μM 2-mercaptoethanol. iNKT cells were enriched using an NK1.1⁺ iNKT cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) following the manufacturer’s instructions [23]. The NKT cell population was >89% pure among all MACS-purified populations. In addition, CD4⁺CD25⁺ Treg cells and CD11c⁺ DCs were isolated from mice using MACS systems (Miltenyi Biotech, Bergisch Gladbach, Germany), following the manufacturer’s instructions. CD4⁺CD25⁺ Treg cells and CD11c⁺ DC population among all MACS-purified populations were >92% and >95% pure, respectively.