Cytotoxic CAR-T cell assay

CAR-T cells, the effector cells, were co-cultured with target cells (B cell lines, myeloid cell lines, or CD19^KO cell lines) at an effector:target cell ratio ranging from 1:1 to 1:10 for 12 h. The LDH release assay (ab102526; Abcam) was used to analyze the lysis of target cells while setting the cell lysate as the positive control (PC) and DMEM as the NC. Briefly, 100 µL of supernatant and 20 µL reaction solution were added to a 96-well plate in the dark, and the absorbance was measured at 590 nm after a 20–30 min reaction. Supernatants were sampled after coculturing for 12 h and an enzyme-linked immunosorbent assay was performed with human interferon gamma (IFN-γ; VAL104; R&D Systems) and an IL-2 kit (VAL110; R&D Systems), according to the manufacturer’s instructions.

The proportion of lysis or specific lysis (%) = [(absorbance of co-culture-NC background)-(auto-release LDH of the CAR-T-NC background)-(auto-release LDH of the target cells-NC background)]/[(maximum LDH release of target cells-volume correcting)-(auto-release LDH of target cells-NC background)] × 100 %.