Mtt cell proliferation assay kit

Cell proliferation was assessed by the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA). HUVECs were seeded in 96-well plates (5000 cells/well) and treated 24 h later with forskolin, IBMX, or 8-bromo-cAMP for an additional 48 h. Cells were then incubated with MTT reagent (10 µl in 100 µl cell culture medium) for 4 h at 37°C, followed by 18 h incubation with 100 µl of crystal dissolving solution at 37°C. Absorbance was measured in 4 different wells at 570 nm. The experiment was repeated twice.

The protective effects of baicalein on Vero cells against Stx were determined. Vero cell culture (0.1 mL) was added to each well of 96-well microtiter plate at 2 × 10⁴ cells per well and cultured for 24 h. One hundred microliters of baicalein at 0.08 and 0.4 mmol/L in PBS were added to each well and incubated for 1 h at 37 °C. Stx preparations (0.1 mL) were added to each well, then further cultured for 48 h at 37 °C in 5% CO₂ incubator. The final concentrations of baicalein in the culture were 0.027 and 0.13 mmol/L. For control, PBS was used instead of the polyphenol. The viability of the Vero cells was determined using the MTT Cell Proliferation Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA), according to the supplier’s instruction.
Three replicates of the treatments were carried out per experiment. The values were reported as an average ± SD of A₅₉₅. The statistical significance of the OD values was calculated by the Student’s T-test.