Wst 8 cell counting kit

A WST-8 Cell Counting Kit (Wako, Osaka, Japan) was used to assess the proliferation of cells grown in 96-well microplates. Prostate cancer cells were seeded in DMEM medium containing 10% fetal bovine serum (FBS) in 96-well plates (5 × 10³ cells/well), and a WST-8 assay was performed as previously described [16 (link)].

Stock solution of MeHgCl (10 mM) (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in Dulbecco’s PBS (Sigma-Aldrich, MO, USA) with l-cysteine (Hg:Cys = 1:1) and kept at − 80 °C until use. The stock was diluted with culture medium just before being added to the cells. NAC (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in FBS-free DMEM, and the pH was adjusted to 7.4 with sodium hydroxide (NaOH).
For cytotoxicity assays, the cells were cultured for 24 h in 96-well plates and incubated with MeHg (0.1–100 µM) for 24 h. Cell toxicity was assayed using the WST-8 Cell Counting kit, according to the manufacturer’s protocol (Wako Pure Chemical Industries, Osaka, Japan). In brief, the cells were washed, and Hank’s balanced salt solution (100 µL) and WST-8 solution (10 µL) were added to each well and kept for incubation at 37 °C in 5% CO₂. The colour was quantified within 1–2 h by a micro-plate spectrophotometer (TriStar LB941, Berthold Technologies, Germany) at 450 nm absorbance. Culture medium was used to adjust the absorbance values of the sample. Mean values and standard errors (SEs) were obtained from the results of four experiments.