Anti phosphos6 clone d57.2.2e

For staining surface markers, cells were washed and blocked in FACS buffer (PBS plus 1% FCS) in the presence of Fc block. Samples were stained with anti-CD8α (clone 53–6.7, BioLegend), anti-CD25 (clone 7D4, eBioscience), anti-CD62L (clone MEL-14, eBioscience), anti-CD69 (clone H1.2F3, eBioscience), anti-Vα2 (clone B20.1, BD Biosciences), anti-CD244 (clone C9.1, BD Biosciences), anti-Ly108 (clone 13G3, BD Biosciences), or anti-CD27 (clone LG.3A10, BioLegend), at 4 C for 30 minutes in FACS buffer, protected from light, followed by fixation with 4% paraformaldehyde (PFA, Electron Microscopy Sciences). For intracellular staining, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) at 4 C for one hour. Samples were then stained with anti-granzyme B (clone GB11, BD Biosciences) for one hour at 4 C, protected from light. For phospho-antibody staining, cells were fixed with 4% paraformaldehyde, methanol-permeabilized at −20 C, and stained for 60 minutes at 4 C with anti-phosphoS6 (clone D57.2.2E, Cell Signaling) in PBS plus 1% Triton X-100 and 0.5% bovine serum albumin (BSA). Data were acquired on either a Calibur1 or LSRII flow cytometer (BD) and analyzed using FlowJo software (Tree Star).