Wst 8

The cells were pre-cultured to early confluency in a 96-well culture dish, then further cultured in the presence or absence of As³⁺, Sb³⁺, Bi³⁺, or ML792 and/or TAK792. The cells were washed twice with HBSS and the viable cell numbers were assayed colorimetrically by WST-8 (Wako) using a microplate reader (POLARstar OPTIMA, BMG Labtech, Offenburg, Germany).

B16F10-NFκB-Luc2 cells were plated at a final concentration of 2 × 10⁴ cells/well in a 96-well plate. After 24 h of incubation, the cells were pretreated with 50 μg/mL of extract or compounds and further incubated for 24 h. Subsequently, 10 µL of WST-8 (FUJIFILM Wako Pure Chemical Corporation) solution was added to the cells, which were incubated for an additional 1 h in a humidified atmosphere (37 °C and 5% CO₂) to allow for the formation of formazan dye and to increase sensitivity. Then, the absorbance was measured with a microplate reader (Sunrise™; Tecan Group Ltd., Männedorf, Switzerland) at wavelengths of 450/620 nm. Cell viability was determined based on the absorbance of the soluble formazan dye generated by the living cells. The same method was followed to determine the viability of B16F10-NFκB-luc2 cells treated with selected extracts in a concentration-dependent manner. The WST-8 solution was added and used according to the manufacturer’s instructions. The viability of the treated cells was calculated as a percentage [13 ,14 (link)].

We assessed the effects of telmisartan on cell viability using water‐soluble tetrazolium (WST)‐8 (Wako Chemicals, Osaka, Japan), a cell proliferation reagent 32. Briefly, aliquots of 1 × 10⁵ cells·mL⁻¹ (cell lines) or 1 × 10⁶ cells·mL⁻¹ (PBMCs) were incubated in 96‐well plates in the absence or presence of telmisartan. After cultivation, we added 10 μL of WST‐8 for 2 h and measured absorbance at 450 nm (A₄₅₀) using an Infinite^® 200 PRO (Tecan, Männedorf, Switzerland). Measurement of mitochondrial dehydrogenase cleavage of WST‐8 to formazan dye provided indication of relative cell viability levels.

CAM, H₂O₂ (30%), the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium) assay system, GSH, and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GSH reductase (from yeast) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were from Oriental Yeast Co., Ltd. (Tokyo, Japan). Buthionine sulfoximine (BSO) and mouse anti-β-actin monoclonal antibody were from Sigma Chemical Co. (St. Louis, MO). A human IL-8 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bender Med Systems (Vienna, Austria). Rabbit polyclonal antibody against γ-GCS was from Neomarkers (Fremont, CA). The nuclear extraction and nuclear transcription factor ELISA kits were from Panomics (Fremont, CA). Cell culture media were obtained from Lonza (Walkersville, MD). All other chemicals used were of reagent grade.