An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids

Published: May 31, 2024

Abstract

Source: Dillard, P., et al. A Spheroid Killing Assay by CAR T Cells. J. Vis. Exp. (2018)

This video demonstrates an in vitro assay evaluating the cytotoxic effects of CAR T cells on 3D tumor spheroids expressing fluorescent proteins. This assessment utilizes the Annexin V red fluorescent probe, which binds to exposed phosphatidylserine residues on the outer leaflet of apoptotic cancer cells after interactions with CAR T cells. The red fluorescence emitted by the probe aids in distinguishing these cells from healthy ones emitting green fluorescence.

Protocol

1. 3D Tumor Spheroid Killing Assay After 6 days or once spheroids reach the desired size, remove the plate from the incubator. Using a multichannel pipette, gently remove 100 µL/well of complete RMPI 1640 medium from the spheroid plates.​ For this step, angle the tips towards the inside wall of the 96-well plate, avoiding contact with the bottom of the well in order to minimize disturbance of the spheroids. The remaining volume should be around 100 &#18…

開示

The authors have nothing to disclose.

Materials

Dulbecco's Phosphate Buffered Saline SIGMA-ALDRICH D8537-500ML Lot Number: RNBG7037
RPMI 1640 med L-glutamin, 10 x 500 ml Life Technology (Gibco) 21875-091 Lot Number: 1926384
Trypan Blue Solution, 0.4% Thermo Fischer 15250061 Lot Number: 1886513
96 well plate, round bottom VWR 734-1797 Lot Number: 33117036
Dynabeads Human T-Activator CD3/CD28 Thermo Fischer 11132D
IncuCyte Annexin V Red Reagent Essen Bioscience 4641 Lot Number: 17A1025-122117
15 ml tubes VWR 734-1867 Lot Number: 19317044
anti-human CD19-PE BD Biosciences 555413 Lot Number: 4016990
RRID: AB_395813
HCT 116 Colorectal Carcinoma Line ATCC CCL-247

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記事を引用
An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids. J. Vis. Exp. (Pending Publication), e22253, doi: (2024).

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