In Vitro Cytotoxicity Assay

Adherent tumor cell lines were plated at 1.25 × 10⁴ cells per well (OVCAR-3) or 2.5 × 10⁴ cells per well (LOX-IMVI) in a 96-well flat-bottomed tissue-culture-treated plate and allowed to rest overnight. CAR-T cell cultures (a mix of NGFR⁺ and non-transduced T cells) were added at various effector:target (E:T) ratios (from 0.25:1 to 8:1) in triplicate, and co-cultures were incubated for 6 h at 37°C. To resolve cytotoxicity, wells were washed 3× with warmed PBS to remove any non-adherent cells, and 100 μL 10% solution of alamarBlue Cell Viability Reagent (Life Technologies) in T cell media was added. After a 3- to 4-h incubation at 37°C, color change was measured by fluorescence (excitation, 530 nm; emission, 595 nm) on a Synergy plate reader (BioTek, Winooski, VT, USA). Tumor cell viability was calculated as the loss of fluorescence in experimental wells compared to untreated target cells.