Anti cd8α clone 53 6

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).

To evaluate the proliferative capacity of cells, splenocytes were stained with 1μM Cell Trace Violet (CTV, Life Technologies) in PBS at 37 C for 10 minutes. Stained cells were washed 3 times with complete media and then stimulated in the presence of OVA_257-264. Cells were collected at indicated time points, stained with anti-CD8α (clone 53–6.7, BioLegend) and evaluated via flow cytometry.

For degranulation assays (30 (link)), activated CTLs were stimulated in plates coated with anti-CD3ε (BioXCell) or mixed at a 1:1 ratio with peptide-pulsed or non-pulsed targets at 37°C in the presence of anti-CD107a-PE-labeled antibody (clone 1D4B, BioLegend). Upon degranulation, CD107a is exposed on the cell surface, allowing binding and subsequent internalization of the anti-CD107a-PE. At indicated time points, plates were placed on ice and cells transferred into cold PBS, stained with anti-CD8α (clone 53–6.7, BioLegend) antibody, and analyzed via flow cytometry. For analysis, the percent of PE⁺ cells in the CD8⁺ gate indicates cells that have degranulated, having been exposed to anti-CD107a during the assay.