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 …

Offenlegungen

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

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Diesen Artikel zitieren
A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells. J. Vis. Exp. (Pending Publication), e21917, doi: (2024).

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