Realease cd8 microbead kit

First, CD8⁺ TILs were collected by magnetic cell sorting using the REAlease CD8 MicroBead kit by positive selection (Miltenyi Biotec) according to the manufacturer’s instructions. After cell sorting, CD8⁺ TILs were expanded by stimulation with Phytohemagglutinin-L (Sigma-Aldrich) and 150 U/mL human rIL-2 in the presence of irradiated allogeneic feeder cells (PBMC and B-EBV cells), as previously described.^{22 (link)} Second, for extensive phenotypic and functional characterizations, CD94/NKG2A negative and positive CD8⁺ TILs were sorted using anti-PE MicroBeads UltraPure Kit (Miltenyi Biotec). Briefly, CD8⁺ TILs were stained with PE-conjugated anti-CD94 mAb (BD Biosciences, HP-3D9, RRID:AB_396201) for 30 minutes at 4°C in PBS 0.1% BSA (Sigma-Aldrich) and then washed twice in PBS 2 mM EDTA 0.5% BSA. After centrifugation at 300 g for 10 min, cells were resuspended in anti-PE microBeads Ultrapure, incubated for 15 min at 4°C and washed again. Finally, magnetic cell sorting was performed on MACS LS columns with a MACS separator (Miltenyi Biotec). The purity of each subpopulation was checked before and after cell sorting and routinely by flow cytometry. Four days later, sorted cells were amplified in culture as described above.