Celltiter 96 aqueous one solution cell proliferation mts

1 × 10⁴ cells were cultured in a 96-well culture plate and transfected with 1 μg of each NFIX shRNA in the respective wells and incubated for 48 h. CellTiter 96^®AQueous One Solution Cell Proliferation (MTS) (20 μl) (Promega, United States) was added to each well and incubated for 4 h before readings were obtained using Varioskan Flash (Thermo Fisher Scientific) at 490 nm. The experiment was conducted in triplicate.

CellTiter 96® AQueous One Solution Cell Proliferation MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay kit (Promega, Madison, WI, USA) was used to evaluate the cytotoxicity of the three flavonoids according to the manufacturer’s instruction. Briefly, RD or Vero cells were seeded overnight in 96 well plates with a density of 2 × 10⁵ cells/mL. Cells were then treated with various concentrations of silymarin (6.25, 12.5, 25, 50,100 and 200 μg/mL), baicalein (1.68, 3.37, 6.75, 13.5, 27, 54 and 108, μg/mL) and baicalin (2.78, 5.57, 11.15, 22.31, 44.63 and 89.26 μg/mL) prepared in maintenance media. RD or Vero cells without test compounds served as a negative control. The plate was further incubated for 24 h at 37 °C. An aliquot of 20 μL of MTS solution was added to each well and the plate was incubated for 1 h at 37 °C. The absorbance was measured at 490 nm with an Infinite 200 Pro Multiplate Reader (Tecan, Männdedorf, Switzerland) (Supplementary Figure S1). The half-maximal cytotoxic concentration (CC₅₀) and 10% cytotoxic concentration (CC₁₀) for each flavonoid were determined using GraphPad Prism software, version 7 (GraphPad Software, Calif, USA).