Tumor infiltrating lymphocytes (TILs) isolation and analysis

CTLs adoptively transferred Rag2^−/− mice or tumor-bearing C57BL/6 mice were anesthetized and sacrificed, tumor tissues were dissected and cut into pieces and digested in RPMI 1640 medium containing collagenase VI (210 U/mL), DNase I (100 U/mL) and hyaluronidase (0.5 mg/mL) for 30 min at 37 °C. The dissociated cells were passed through a 70 µm strainer. The filtered cells were centrifuged at 50 ×g for 1 min. Then the supernatant was removed to a new tube to centrifuge at 1000 ×g for 10 min. Resuspended cells for density gradient centrifugation with 40% Percoll and 70% Percoll. Harvest the interphase of gradient and spin at 1000 ×g for 5 min. The isolated tumor infiltrated lymphocytes were then used in the subsequent experiments. To measure the cytokine production of isolated TILs, the cells were stimulated with 50 ng/mL PMA, 1 µmol/L ionomycin and 5 µg/mL BFA for 4 h at 37 °C.