Vero cells

The effects of baicalein (NAMIKI SHOJI Co., Ltd., Tokyo, Japan) on the cytotoxicity of Stx were determined according to the previously reported method [16 (link)]. Vero cells were purchased from Cell Bank (RIKEN BioResource Center, Tsukuba, Ibaraki, Japan). The cell viability was determined using the MTT Cell Proliferation Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA), following the supplier’s instruction. Medium was removed and fresh 5% FBS-MEM-E medium (10 µL) was added to the well. An amount of 12 mmol/L MTT stock solution (10 µL) was added to each well. The medium was removed after incubation at 37 °C. To resolve the MTT formazan crystals formed, 100 µL of Cell-Based Assay Buffer was added to the well. Medium alone was used as a negative control. Absorbance at 595 nm was measured using Microplate Reader Model 680 (Bio-Rad Laboratories Inc., Hercules, CA, USA). A value OD at 595 nm was used to show cell viability.
To determine the inhibitory activity of baicalein, various concentrations of the Stx preparations were incubated with baicalein before addition to the Vero cell culture. The final concentration of baicalein in the culture was 0.027 and 0.13 mmol/L. Three replicates of the treatments were carried out per experiment and the values were shown as an average ± SD of A₅₉₅. The statistical significance of the OD values was calculated by the Student’s t-test.

293T cells (human embryonic kidney cell line) and Vero cells (African green monkey kidney cell line) were obtained from the RIKEN BioResource Center Cell Bank. Calu-3 cells (human bronchial epithelial cell line), MRC5 cells (human fetal lung fibroblast cell line) and HCT8 cells (human rectal adenocarcinoma cell line) were obtained from the American Type Culture Collection (ATCC). THP-1 cells and Vero/TMPRSS2 cells⁵³ (link) were obtained from the Japanese Collection of Research Bioresources Cell Bank. 293T, Calu-3 and MRC5 cells were maintained in Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS). HCT8 and THP-1 cells were maintained in RPMI-1640 medium with 10% FCS. Vero cells and Vero/TMPRSS2 cells were maintained in DMEM containing 10% FCS. 1 mg/mL G418 (Invivogen) was added to the growth medium for Vero/TMPRSS2 cells. THP-1-dual reporter cells, THP-1-dual KO-RIG-I cells and THP-1-dual KO-MDA5 cells were purchased from InvivoGen and maintained according to the manufacturer’s instructions.