A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

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 …

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