Cytotoxicity assay

Long-term tumor cell killing was determined by xCELLigence, an impedance-based real-time cytotoxicity assay (RTCA). Background impedance of RTCA E-plates and cell culture media was first measured. Tumor cells (U251 or U87) were seeded on untreated RTCA E-plates (20,000 cells per well). For the glioblastoma primary lines, wells were coated with 1 μg/cm² laminin and 40,000 cells were seeded in each well for 14–18 hours for cells to adhere. EphA3 CAR T cells were added at the same ratio as targets. Changes in impedance were reported every 30 min as Cell Index. Normalized CI values were used to calculate the percentage of cytolysis using the following: Percentage of cytolysis = ((Cell Index _{no effectors} – Cell Index _effector)/Cell Index _{no effectors}) × 100. EC 50 values were calculated from the sigmoidal dose-response curve calculated with the GraphPad Prism V.9.4.0 software (GraphPad software).