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 …
Disclosures
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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Cite This Article
An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids. J. Vis. Exp. (Pending Publication), e22253, doi: (2024).