Anti cd244

Splenocytes were isolated and prepared as a single-cell suspension. 3×10⁶ WT and 2B4KO OT-I T cells, respectively, were resuspended in 1.5 mL of complete media (glucose-free RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamine), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, and 0.5 mM 2-mercaptoethanol) in a 24-well flat-bottomed plate at 37°C in a 5% CO₂, humidified atmosphere. A D-glucose solution of 200g/L (Gibco by Life Technologies) was titered into the glucose-free complete media and diluted on a half-log scale. Cells were then stimulated with SIINFEKL N4 peptide at 1 nM for 5 days. After 5 days in culture cells were harvested and stained with anti-CD8 (BD Horizon), anti-Thy1.1 (BD Biosciences or BioLegend), anti-CD244 (eBioSciences), anti-Va2 (BD), anti-CD127 (BioLegend), anti-CD62L (BD), anti-KLRG-1, anti-Vb5 (BD), anti-CD44 (BD), 7AAD (BD) and AnnexinV (BioLegend), anti-IL-2 (BD), anti-IFN-g (BD) as described by the manufacturer. As described above, samples were analyzed on an LSRII flow cytometer (BD Biosciences) and data was analyzed using FlowJo 9 software (Treestar, San Carlos, CA) and Prism 6 software (GraphPad Software Inc.).