After co-culturing CAR expressing jurkats with CFSE-labeled tumor cells, gently invert the plate on a paper towel and tap to discard the supernatants containing CAR-J. Using a multichannel pipette, add 100 microliters of FluoroBrite DMEM media containing propidium iodine, or PI, into the wells without disturbing the adhered tumor cells. Then add 20 microliters of 20%Triton X to the first well of each tumor type, serving as a total dead control.
Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 20 minutes before imaging. After fluorescent imaging, use one of the tumor-only wells of cells to calibrate the green fluorescent channel. To exclude cells at the edge of the wells decrease the well mask to 98%Identify CFSE-labeled tumor cells on the green fluorescent channel and adjust the fluorescence intensity threshold value to capture all the cells in the well.
Set the minimum cell diameter to 25 micrometers to exclude debris detected in the CFSE channel. Then enable separate touching objects to identify individual cells. Next set gates to define live versus dead cell populations.
Select the CFSE-labeled cells and generate a histogram of counts on the y-axis versus mean PI intensity on the x-axis. Adjust the x-axis value to better visualize the cells and draw a splitter to differentiate between low PI stained cells and high PI stained cells. Label the low PI stained cells as live cells.
Next, set another histogram on live cells with area on the x-axis and counts on the y-axis. Label the non-debris cells as big live cells. Then draw another splitter using the tumor only well to capture cells and filter out debris.
After running the analysis on the entire plate, export the spreadsheet containing the numbers. Plot the counts of big cells to determine the number of live CFSE-labeled tumor cells remaining in the well after exposure to CAR-J. Finally perform statistical analysis using one-way ANOVA.
The cytotoxicity of CAR-J1 increased with a higher effector to tumor ratio and more than 50%killing was observed at an effector to tumor ratio of four to one. EGFR-targeting CAR constructs with modified hinge domains showed antigen-specific killing against EGFR expressing MDA-MB-231 cells, and no significant killing was observed against EGFR knockout cells. CAR constructs were also expressed on PBMC derived CD3 T cells.
However, there was no expression of CD8 hinge-containing EGFR-CAR. The IgG short showed the least cytotoxic potency against MDA-MB-231 cells compared to the IgG long and IgG medium.
