2.8. CAR-T cytotoxicity assay

Untransduced-T or CD19CAR-T cells were pre-stained with CellTrace Violet (CTV) dye (Invitrogen, Carlsbad, CA, USA) and then resuspended to an appropriate density with T lymphocyte medium, and co-cultured with target cells at various E:T ratios (10:1, 5:1, 1:1, 1:5, and 1:10) in a 96-well plate for 24 h. The 7-AAD Viability Staining Solution was used to stain dead cells and analyzed by flow cytometry. The equation used to calculate cytotoxicity percentage was 100 × (target cell death – spontaneous cell death)/(100 – spontaneous cell death).