2.4. MTT assays for cell viability

For in vitro cell viability measurement, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assays were performed in the four human lung carcinoma cell lines (GLC‐82, A549, NCI‐H460, and NCI‐H1299), as well as human lung fibroblasts. Twenty‐four hours before treatment, cells were seeded in a 96‐well plate and grown to reach approximately 80% confluence. Cells were infected with recombinant CVB3 at MOI of 6.67 × 10⁻⁶ 6.67 × 10⁻⁵, 6.67 × 10⁻⁴, 6.67 × 10⁻³, 6.67 × 10⁻², 6.67 × 10⁻¹, 6.67 × 10⁰, 6.67 × 10¹, 6.67 × 10²; or exposed to normal saline (NS) or cisplatin (CIS) as the negative and positive controls, respectively. After 72 hours, MTT assays were conducted in accordance with the manufacturer's protocol (VWR Life Sciences Amresco, Radnor, PA, USA). Briefly, the cell culture medium was replaced with 200 μL of MTT (0.5 mg/mL) in cell culture medium containing 10% fetal bovine serum, and cells continued to incubate at 37°C for 1 hour. Supernatants in each group were removed, and 200 μL of dimethyl sulfoxide was added in each well to solubilize the MTT dye. Absorbance was read at 570 nm on a microplate reader. Each condition was tested with 6 replicates and all assays were performed in triplicate. A half‐maximal inhibitory concentration (IC₅₀) was calculated in each group. The inhibition rate (IR) was calculated using the following formula: IR = [Absorbance (NS) – Absorbance (CVB3 oncolytic virus)/Absorbance (NS)] × 100%.