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 µL.
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.
Add 100 µL/well of the 1:200 Annexin V red solution.
Transfer the plate to an incubator (37°C, 5% CO2, 95% humidity) for 15 min.
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.
Count cells using Trypan blue exclusion on a compatible cell counter.
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.
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.
Transfer the plate back to the automated imaging apparatus inside an incubator (37 °C, 5% CO2, 95% humidity).
Log into the acquisition software,Select Schedule To Acquire.
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.
Set the desired starting time (at least 1 h after incubation in the automated imaging apparatus). Select Save schedule scans.