- Make sure you have enough activated (for at least 3 days), healthy (>50% viability), and cytotoxic (CD8) T cells in culture before starting
- Make sure you have access to a flow-cytometer after the co-culture is done
- When in doubt, use 24-well plates for the co-culture
- Make sure the cell line expresses the target protein (for CAR) or presents the relevant peptide (for TCRs) up-front
- Make sure the cell line can grow and sustain viability in T cell media throughout the co-culture
- Make sure the final T cell concentration doesn't go higher than 2 million per mL since this can cause stress on the T cells and the cell line
- This protocol assumes the assay is carried out at 8:1 T-cell:Cell-line ratio. Please scale the numbers up if you would like to assay at a different scale/ratio
- When in doubt, use OT-I CD8 T cells against MC38s that are pulsed with the SIINFEKL peptide as a positive control
- When in doubt, use OT-I CD8 T cells against MC38s that are NOT pulsed with the SIINFEKL peptide as a negative control
- This protocol assumes the T cells and the cancer cells are of mouse origin. If you are using a different organism or the channels are not appropriate for your flow-cytometer, please customize your antibodies accordingly