This study investigates the efficacy of CAR T cells in targeting B cell lymphoma tumor cells. The experimental setup involves co-culturing CAR T cells with tumor cells and measuring the resulting cytotoxic effects through bioluminescence.
Take a multiwell plate containing B cell lymphoma tumor cells expressing a B cell-specific surface antigen and a cytoplasmic luciferase enzyme.
Introduce CAR T cells into selected wells and incubate. The cells express chimeric antigen receptors, or CARs, comprising an extracellular domain targeting the tumor antigen and intracellular signaling domains.
CAR binds to the tumor antigen, inducing activation of the CAR T cells.
The activated cells release cytokines that promote the cells' anti-tumor activity, triggering the release of toxins.
The toxins enter the tumor cells and induce cell death.
Spin down the cells and isolate the supernatant. Quantify the cytokines to assess CAR T cell activity.
Resuspend the cells in a buffer containing luciferin, a bioluminescent substrate.
Upon entry, luciferin is oxidized by luciferase in living tumor cells, emitting light.
Measure the bioluminescence to identify a decreased signal in the treated tumor cells, confirming CAR T cell-mediated cytotoxicity.
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.
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