Na ve mouse cd8 t cell isolation kit

Spleens were isolated from OT-I mouse strain and placed in ice-cold wash buffer (2% FBS in PBS). Spleens were dissociated mechanically, passed through a 100 μm filter, and incubated with ACK Lysis Buffer (Lonza) for 2 minutes at room temperature to lyse RBCs. The resulting cell suspension was washed with wash buffer, filtered through a 40 μm filter and then resuspended in MACS Buffer (0.5% BSA and 2 μM EDTA in PBS). Naive CD8⁺ T cells were isolated using naïve mouse CD8 T cell isolation kit from Miltenyi (130-104-075). Naive CD8⁺ T cells were counted and then resuspended in cRPMI (RPMI-1640 with 10% FBS, 2mM L-Glutamine, 1% HEPES, 1% 100 nM NaPyruvate, 1% NEAA, 100U Pen/Strep and .05 μM β-mercaptoethanol) to a final concentration of 1 × 10⁶ cells/ml. The cells were then plated into a 12 well plate at a concentration of 1 × 10⁶ cells per well. These CD8 T cells were expanded in-vitro using a previously published protocol ^{63 (link)}. In brief, cells were expanded for 3 days in cRPMI media supplemented with 10 ng/mL OVA (Anaspec AS-60193-1), 5 ng/mL IL7 and 5 ng/mL IL15. Cytokines were purchased from Peprotech. After 3 days of in-vitro expansion CD8 T cells were used for co-culture experiments.