Anti eomes dan11mag

The following cell surface antibodies (purchased from eBioscience, San Diego, CA) and their respective isotype controls were used: anti-CD8α (53-6.7), anti-CD27 (LG.7F9), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD107a (1D4B), anti-Thy1.1 (HIS51) anti-PD-1 (J43) and anti-OX40 (OX86). Anti-Vβ8.3 TCR (1B3.3) was purchased from BD Biosciences (Oxford, UK). Direct intracellular staining was carried out using anti-perforin (OMAK-D), anti-T-bet (4B10) and anti-Eomes (Dan11mag) and isotype controls, purchased from eBioscience, San Diego, CA or anti-granzyme B (GRB05) from Life Technologies, UK. Following brief peptide re-stimulation, intracellular staining was performed using anti-IFN-γ (XMG1.2) or anti-TNF-α (MP6-XT22) together with the appropriate isotype controls (BD Biosciences, Oxford, UK). Intra-nuclear staining for BrdU was carried out using anti-BrdU-APC flow kit (BD Biosciences, Oxford, UK), according to the manufacturer's instructions. To detect CD107a cells were restimulated for 4 hours in the presence or absence of 1μM UTY peptide with Golgi-Stop (BD Pharmingen) in the presence of anti-CD107a or isotype control. Cells were then re-surface stained with anti-CD107a or isotype. Flow cytometric analysis was performed on a LSRFortessa or FACs Canto II (BD Biosciences) and cell counting performed on a Coulter Counter (Beckman Coulter).