Mouse il 15 il 15ra

Thymocytes from WT mice were enriched for Vα14i NKT cells by magnetic depletion using biotinylated antibodies against CD8α, CD19, CD24 and TER-119 (BD Biosciences and eBioscience) together with EasySep magnets and protocols and reagents from StemCell Technologies. Cells were then stained with αGalCer-loaded CD1d tetramers, together with anti-TCRβ antibodies, in staining buffer containing 1 μg/mL streptavidin. Tetramer-positive, TCRβ+ cells were isolated using a FACSAria cell sorter (BD Biosciences). Sorted Vα14i NKT thymocytes were then cultured for 48 hours at 10⁶/mL in complete RPMI media (supplemented with 10% FBS, 50 μM β-mercaptoethanol, 1X penicillin/streptomycin/glutamine mix, and 20 mM Hepes) on a plate coated with anti-TCRb antibody together with soluble anti-CD28. Cells were then maintained by culture in complete RPMI media with 10ng/mL mouse IL-15/IL-15Ra (eBioscience) for 5 days before being used in experiments.

Thymocytes from WT mice were enriched for Vα14i NKT cells by magnetic depletion using biotinylated antibodies against CD8α, CD19, CD24, and TER-119 (BD Biosciences and eBioscience) together with EasySep magnets and protocols and reagents from StemCell Technologies. Cells were then stained with αGalCer-loaded CD1d tetramers, together with anti-TCRβ antibodies, in staining buffer containing 1 μg/ml streptavidin. Tetramer-positive, TCRβ⁺ cells were isolated using a FACSAria cell sorter (BD Biosciences). Sorted Vα14i NKT thymocytes were then cultured for 48 h at 10⁶/ml in complete RPMI media on a plate coated with anti-TCRβ antibody together with soluble anti-CD28. Cells were then maintained by culture in complete RPMI media with 10 ng/ml mouse IL-15/IL15Ra (eBioscience) for 5 days before being used in experiments.