An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

First, seed 10,000 syngeneic target CD19-positive tumor cells with or without luciferase expression, and 100 microliters of TCM into each well of a 96-well U-bottom tissue culture plate. Add 10,000 CD19 CAR T cells and 100 microliters of TCM to each well.

Set up the controls as outlined in the text protocol. Co-culture the cells at 37 degrees Celsius with 5% carbon dioxide for 16 to 24 hours. After this, centrifuge the plate at 500 times g for five minutes. Collect the supernatant for further interferon-gamma and interleukin-12 p70 ELISA analysis.

Resuspend the cell pellets in 100 microliters of PBS containing luciferin, and incubate at 37 degrees Celsius for 10 minutes. Then, use a luminometer to measure the luminescence from each well.