51Cr Release Cytotoxicity Assay for murine CAR T cells

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.

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)

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

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

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.

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.

10.

Acquire data on the plate scintillation counter per respective protocol.

Abstract

Steps

Labeling target cells with 51Cr (day 0)

Processing effector cells (day 1)

Reading scintillation plate

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