Aug 21, 2023
51Cr Release Cytotoxicity Assay for murine CAR T cells
- Tamer B Shabaneh1,
- Andrew R Stevens1
- 1Fred Hutching Cancer Center
Protocol Citation: Tamer B Shabaneh, Andrew R Stevens 2023. 51Cr Release Cytotoxicity Assay for murine CAR T cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmm2ol5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 20, 2023
Last Modified: August 21, 2023
Protocol Integer ID: 86711
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").
Labeling target cells with 51Cr (day 0)
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.
Note
Overnight chromium incorporation is often sufficient, but the duration may vary depending on the cell line. If the positive control (soap-killed cells) endpoint values are inadequate, the length of time can be adjusted.
Processing effector cells (day 1)
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.
Note
mTCM is prepared by combining the following:
1000 mL RPMI1640 (with 25mM HEPES)
100 mL FBS (heat inactivated)
10 mL Sodium pyruvate (1mM)
1 mL HEPES (1M)
10 mL Pen/Strep
100 mL b-mercaptoethanol 0.5M
Filter with 0.22mm
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.
Note
This would assess cell killing for one CAR T cell culture against one target. Scale up as needed by seeding additional rows.
Example layout:
Desired E:T ratios are 30:1 [3x104:1x103], 10:1, ~3:1, and ~1:1
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.
Note
Non-enzymatic dissociation is a critical step for preserving cell-surface antigen.
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
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.