Fig. 3.

Bioorthogonal targeting selectively and effectively boosted cytotoxicity of CAR-T for tumor cells. (a) Time-lapse live microscopy was used to measure CAR-T cell cytotoxicity against target cells in the presence of PI (100 μM). Images were acquired every 30 min, and displayed the overlays of the anti-CD3-FITC antibody (green), PI (red), and bright field channels.(b) The apoptosis of Raji cells were analyzed by flow cytometry using PI at 3 h post administration of CAR-T cells. (c) LDH release analysis of CAR-T cell killing activity to Raji cells. (d–e) Flow cytometry analysis evaluated the selective cytotoxicity of azido CAR-T cells in mixed Raji cells. (f-h) The killing activity of CAR T cells with or without bioorthogonal targeting was analyzed using the 3D tumor spheroid model. The death rate of tumor cells in 3D tumor spheroids was separately analyzed using confocal imaging and flow cytometry at 24 h post-incubation.