51Cr Release Cytotoxicity Assay for murine CAR T cells

Tamer B Shabaneh, Andrew R Stevens

Published: 2023-08-21 DOI: 10.17504/protocols.io.n92ldmm2ol5b/v1

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)

1.

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.

2.

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)

3.

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/Strep100 mL             b-mercaptoethanol 0.5MFilter with 0.22mm

4.

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.

4.1.

Example layout:

Desired E:T ratios are 30:1 [3x104:1x103], 10:1, ~3:1, and ~1:1
Desired E:T ratios are 30:1 [3x104:1x103], 10:1, ~3:1, and ~1:1
5.

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.

5.1.

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.

6.

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. 

7.

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.

8.

Centrifuge plate at 800rpm for 3mins; incubate at 37C for 24 hours.

Reading scintillation plate

9.

Harvest 30uL supernatant from each plate; carefully transfer onto respective Luma plate wells.

Dry the plate completely for ~24 hours.

10.

Acquire data on the plate scintillation counter per respective protocol.

推荐阅读

Nature Protocols
Protocols IO
Current Protocols
扫码咨询