A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells
Published: January 31, 2024
Abstract
Source: Xi, B. et al., A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells. J. Vis. Exp. (2019)
This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic capabilities.
Protocol
1. Generation of CAR-encoding lentivirus NOTE: Once the specific chimeric antigen receptor (CAR) T-cell plasmid construction is completed (CD47 and others), lentiviral CARs are generated by the standard procedure, using 293 FT cells, a lentiviral packaging mix, and transfection agents (see the Table of Materials) as described. Subsequently, use a quantitative reverse transcription polymerase chain reaction (RT-PCR) kit and a thermal cycler …
Divulgations
The authors have nothing to disclose.
Materials
| 7-AAD | Biolegend | 420404 | |
| Anti-CD40, liquid tumor killing assay kit | ACEA Biosciences | 8100005 | |
| anti-human F(ab')2 | Jackson Immunoresearch laboratories. | 109-116-088 | |
| APC anti-CD3 | Biolegend | 317318 | |
| Assay medium RPMI1640 | life technologies.Corp | 11875-093 | |
| CAR-T cell frozen solution CryostorCS10 | Stemcell technologies | #07930 | |
| CAR-T cell medium from ProMab | AIM-V+300IFU/ml IL-2 | 12055-091 | |
| CD3/CD28 coated microbeads, Dynabeads | Thermofisher | 11131D | |
| DMEM | GElifesciences.com | SH30243.02 | |
| FACS buffer | Promab made | ||
| FBS | Lonza.com | 14-503F | |
| HEK293FT | Thermo Fisher | R70007 | |
| INFg ELISA kit | Thermo Fisher | ||
| Lentiviral Packaging Mix | System Biosciences | VP100 | |
| Lenti-X quantitative RT-PCR titration kit (Clontech) | Takara | 631235 | |
| Promab medium for target cells | Varied with cell lines | ||
| Real time Cellular Analyer | ACEA Biosciences | ||
| Thermal cycler | Thermo Fisher | ||
| Transduction enhance agent, Virus Transduction Enhancer (Alstem) | Transplus, Alstem | V020 | |
| Transfection dilution solution, Opti-MEM | Thermo Fisher | ||
| Transfection reagent, NanoFect reagent | Alstem | NF100 |
Tags
Citer Cet Article
A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells. J. Vis. Exp. (Pending Publication), e21917, doi: (2024).