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

  1. 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.
    1. 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 µL.
  2. Prepare a 1:200 solution of Annexin V red by mixing 50 µL of Annexin V red with 9.95 mL of complete RPMI 1640 medium.
  3. Add 100 µL/well of the 1:200 Annexin V red solution.
  4. Transfer the plate to an incubator (37°C, 5% CO2, 95% humidity) for 15 min.
  5. Harvest the transduced CAR CD19 T cells in a 15 mL tube and centrifuge them at 500 x g for 5 min. Remove the supernatant using a pipette and resuspend by pipetting up and down several times with 2 mL of complete RPM1 1640 medium.
  6. Count cells using Trypan blue exclusion on a compatible cell counter.
  7. Centrifuge cell suspension at 500 x g for 5 min. Remove supernatant using a pipette and resuspend in RPMI medium to obtain 2 x 105 cells/mL.
  8. Transfer the cell suspension to a sterile reservoir and dispense 100 µL/well into a 96-well round bottom spheroid plate using a multichannel pipette.
  9. Transfer the plate back to the automated imaging apparatus inside an incubator (37 °C, 5% CO2, 95% humidity).
  10. Log into the acquisition software,Select Schedule To Acquire.
    1. Right-click on the Scan timeline and select Edit Timeline. Right-click on the scan group and delete it. Right-click on the timeline and select Set Selected Scan Group Interval option and set Add scans every to 1.5 h and For a total of to 24 h.
    2. Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus). Select Save schedule scans.

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

タグ

This article has been published
Video Coming Soon
Keep me updated:

.

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).

View Video