51Cr Release Cytotoxicity Assay for murine CAR T cells
Tamer B Shabaneh, Andrew R Stevens
Abstract
A standard cell-based assay to assess the function of murine CAR T cells, which we regularly perform at the end of the process of generating CAR T cells (see "Retroviral transduction of primary murine CD8 T cells").
Steps
Labeling target cells with 51Cr (day 0)
For each adherent tumor cell line, plate 5x105 cells / 3mL (previously growing in log phase) in one 6-well-plate well; Incubate cells for ~3hr to rest.
In the Hot Lab , label the target cells with 50 μL 51Cr (vial <1wk old), 100μL (1-2wk old), or 150μL (>2wk old). Place in Hot Lab incubator overnight.
Processing effector cells (day 1)
From each transduced T cell culture, remove 9x105 cells, wash with fresh mouse T cell media (mTCM) for 6min at 400rcf, and resuspend in 3mL mTCM.
In triplicates, transfer 150μL (4.5x104 effector cells) to U-bottom 96-well-plate wells (in columns 1, 2, 3). Pipet mTCM (100uL/wel) into the remainder of the row (4-12); prepare 3 serial dilutions (1:3) by pipetting 50uL to the respective 100uL volumes.
Begin processing the target cells. To remove extracellular 51Cr, briefly wash the adherent lines with 5mL Cell Dissociation Buffer (0.5mM EDTA in PBS). Repeat the wash. Dispose the 51Cr-containing media appropriately.
To dissociate target cells, add 3mL Cell Dissociation Buffer to each well, incubate at 37C for 10-15 mins. Add 3mL mTCM; centrifuge at 400rcf for 4min.
Count the cells using a hemocytometer. Adjust volume to 1E4 cells / mL mTCM.
Transfer 100μL (1E3 target cells) to each well on the respective half of the plate already containing 100uL of effector CAR-T cells.
Finally, transfer 100μL target cells to an extra row. Add 100μL of soap (NP40+Trypan blue) to generate a positive control. Add 100μL mTCM to generate a negative control.
Centrifuge plate at 800rpm for 3mins; incubate at 37C for 24 hours.
Reading scintillation plate
Harvest 30uL supernatant from each plate; carefully transfer onto respective Luma plate wells.
Dry the plate completely for ~24 hours.
Acquire data on the plate scintillation counter per respective protocol.