Incucyte killing assay

Tumor cell spheroids (RH30 parent or RH30 IL-8 OE line) were made by resuspending 5000 cells/well in a mixture of R10+0.5% polyvinyl alcohol+0.005% Matrigel (100 µL/well) and centrifuging at 1200 rpm for 10 min in ultra-low attachment U bottom 96 well plates (Corning CoStar). 24 hours later, either mock, B7-H3 CAR or BC2 CAR T cells were added at 10 000 cells/well in 100 µL of R10+100 u/mL IL-2. This mixture contained Annexin V red dye (Sartorius) per the manufacturer’s protocol. The plate was placed inside of an Incucyte S1 live-cell imaging system (Sartorius, Essen Bioscience) and images were captured every 2 hours for 90 hours using a 10× lens. Data were analyzed using the spheroid function in the Incucyte 2022 software (Sartorius, Essen Bioscience). Virtual masks were made to identify the GFP tumor spheroid and the Annexin V red activity for each condition.