Im0280

KGN cells (1500 cells/well) or mGCs (4000 cells/well) were seeded in 96-well plates and allowed to continuously grow. MTT (Solarbio, IM0280) was added to the culture medium each day at the appointed time for incubation for 4 h followed by the addition of 100 µl of dimethyl sulfoxide to replace the medium. Absorbance was recorded at 492 nm with a Multiscan plate reader.

The in vitro oncolysis assay was measured by MTT. 4T1, B16F10, CT26, MC38, Vero, BEAS-2B, LO2 cells were seeded in a 96-well plate at 1 x 10⁴ cells per well and recombinant oncolytic VVs were added at the indicated MOI. After a 72 h incubation, 20 μl of MTT solution (5 mg/ml; Cat# IM0280, Solarbio, Beijing, China) was added to each well and the cells were incubated for 4 h. The supernatants were discarded and 150 μl DMSO was added to each well. The absorbance (A) was tested at a wavelength of 570 nm using microplate reader (Spectra-Max M3, Molecular Devices, USA). The cell viability was calculated according to the following formula:

Caco-2 cell lines were purchased from the cell bank of the Chinese Academy of Sciences. Cells were cultured in high glucose DMEM medium (Hyclone, SH30022.01) containing 10% FBS (fetal bovine serum, Gibco, 10099-141), 1% penicillin/streptomycin (Gibco, C14-15070-063) in 5% CO₂ at 37 °C. Squalene (J&K, Shanghai, China) and 25-hydroxycholesterol-3-sulfate (TargetMol, Shangahi, China) were suspended in DMEM, and then Caco-2 cells were incubated with drugs following the given concentration. Cell viability of Caco-2 post treatment was examined using MTT (Thiazolyl blue tetrazolium bromide, Cat. IM0280, Solarbio Life Science, Beijing, China) method following the manufacture manual.