The cytotoxicity % is calculated by the equation below.
Cytotoxicity % = (Live counts of control – Live counts of treated) / Live counts of control * 100
By labeling the target tumor cells with non-toxic, non-radioactive calcein AM or transfect with GFP, we can monitor the killing of the tumor cells by CAR-T cells. While live target cancer cells will be labeled by a green calcein AM or GFP, the dead cells cannot retain the green dye. Hoechst 33342 is used for stain all cells (both T cells and tumor cells), alternatively, target tumor cells can be stained with membrane bound calcein AM, PI is used for stain the dead cells (both T cells and tumor cells). This staining strategy allows for the discriminate of different cells.