Experimental Protocol
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.
E: T Ratio dependent Cytotoxicity of K562
The example Hoechst 33342, CFSE, PI fluorescent images are the K562 target cells at t = 3 hours
The resulting fluorescent images showed increase in Hoechst+CFSE+PI+ Target cells as E: T ratio increased
