An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells
Published: May 31, 2024
Abstract
Source: Kueberuwa, G., et al. A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells. J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to validate chimeric antigen receptor (CAR) T cell activity. B-cell lymphoma cells expressing CD19 antigen and cytoplasmic luciferase are co-cultured with CD19-specific CAR T cells. CAR binding to CD19 on a tumor induces the CAR T cells to release cytokines and toxins that cause tumor cell cytotoxicity. The CAR T cell activity is evaluated by quantifying the released cytokine and decreased luciferase-mediated bioluminescence in the tumor cells.
Protocol
1. In vitro Validation of CAR T cell Activity Seed syngeneic target CD19+ tumor cells with or without luciferase expression at a density of 1 x 104 cells in 100 μL complete T cell medium (TCM)/well in a 96-well U-bottom tissue culture plate. Add 1 x 104 CD19 CAR T cells/well in a volume of 100 μL/well to achieve an effector to target (E:T) ratio of 1:1. NOTE: E:T ratios should be established for each C…
Representative Results
Figure 1. Validation of CAR T-cell activity. αmCD19 CAR T cells were co-cultured with A20 lymphoma cells modified to express luciferase (1 x 104:1 x 104) for 16 h in a U-bottom 96-well plate. After co-culture, cells were pelleted, and supernatant was collected. A) Cells were re-suspended in PBS and luminometry was used to assess the viability of the tar…
Disclosures
The authors have nothing to disclose.
Materials
| 100X penicillin-streptomycin-glutamine (PSG) | Gibco | 10378016 | |
| Flow cytometer – LSR Fortessa x20 | BD Biosciences | 658222R1 | |
| Foetal Bovine Serum | Gibco | 10270 | |
| IL-12 p70 Mouse Uncoated ELISA Kit | Invitrogen | 88-7121-76 | |
| Ionomycin Calcium Salt | Sigma- Aldrich | I0634 | |
| Luminometer – Lumistart Omega | BMG Labtech | 415-301 | |
| Murine IFN-γ ELISA kit | Diaclone | 861.050.010 | |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma- Aldrich | P8139 | |
| RPMI 1640 Medium | Lonza | BE12-167F | |
| XenoLight D-Luciferin | Perkin Elmer | 122799 |
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Cite This Article
An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells. J. Vis. Exp. (Pending Publication), e22258, doi: (2024).