CD107a and granzyme-B functional assay
A flow cytometry-based analysis of T-cell function was designed to detect CD107a membrane expression and intracellular granzyme B expression. Briefly, PBMCs were stimulated at 37 °C overnight at a concentration of 5 × 105 cells/mL in the presence of 25 ng/mL phorbol myristate acetate (PMA) and 1 µg/mL ionomycin. On the day of coculture, cells were stained with CD107a or its IgG1 k isotype (BD Biosciences). Brefeldin A and GolgiStop were added one hour later. After 72 h, cells were washed once with FACS buffer, then surface stained (4 °C for 30 min), followed by intracellular staining with granzyme B antibodies as mentioned above.