Chimeric Antigen Receptor (CAR) T-Cell-Mediated Cytotoxicity
Measure Chimeric Antigen Receptor (CAR) T-Cell-Mediated Cytotoxicity
- Image and plot killing of target cells by CAR-T cells
- Produce data for measuring percent cytotoxicity
- Capture BF and FL images that demonstrate killing of target cells by CAR-T cells
Measure CAR T-Mediated Cytotoxicity using Tracer and Viability Dyes
Chimeric antigen receptor (CAR-T) therapy focuses on generating a genetically modified human t-cell that can directly bind to, and attack cancer cells. The CAR-T cell is comprised of a tumor-specific, antigen-binding domain that is fused to the genetically engineered activating motif. Once expressed on the cell surface the t-cell can target and attack cancer-specific cells.
By labeling the target tumor cells with non-toxic, non-radioactive calcein AM, 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, the dead cells cannot retain the green dye and therefore will release it into the surrounding media. Alternatively, target tumor cells can be stained with membrane-bound CFSE dye at the start of the experiment and counterstained for viability with PI. This staining strategy allows for the detection of dead (CFSE pos+PI+) target cells and dead (CFSE neg+PI+) CAR-T cells.
Imaging and Quantification of CAR T Mediated Cytotoxicity
Celigo Experimental Protocol
- Target cells (adherent or suspension) were collected and stained with tracer dye CFSE
- Target cells were seeded in the wells of microplates
- CAR T cells were added to the wells at different E:T ratios
- At the endpoint, viability dye (PI) is added to determine the viability of target cells
- The wells were scanned and analyzed using Celigo at the endpoint to measure target cell count and viability
- Endpoint percent cytotoxicity data was generated to show cytotoxicity of target cells
Calculating the Cytotoxicity Percentage
The cytotoxicity % is calculated by the equation below.
Target and Effector Cell Fluorescent and Bright Field Overlay Images
Fluorescent Intensity Gating for Cytotoxicity Percentage