Cytotoxicity of CAR-T cells against HCC cell lines was assessed by RTCA. (A) Transduction efficiency of GPC3 CAR, B7H3 CAR, GPC3-BiTE CAR on T cells was detected by flow cytometry. The right three T cells were stained with the Biotin-Goat Anti-Mouse IgG, F(ab') ₂ fragment specific and the right two T cells were stained with Biotin-Goat Anti-Human IgG, F(ab') ₂ fragment specific. (B and C) The cell index curve of RTCA and cytotoxicity statistical analysis of CAR-T cells against Huh7 cell lines. (D) Transduction efficiency of GPC3 CAR, GPC3-BiTE CAR on T cells was detected by flow cytometry. The T cells were isolated from another donor PBMC. (E and F) The cell index curve and cytotoxicity statistical analysis of CAR-T cells against SK-HEP-1-B7H3 cell lines. Statistical significance was assessed by two-way ANOVA (C and F). Not significant ns, P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.