Wst 1 test kit

Cell viability was assessed with a WST-1 test kit (Roche Diagnostics), which is based on the cleavage of water-soluble tetrazolium salt by mitochondrial dehydrogenases in live cells. SiHa cells were seeded in 96-well plates and cultured for 24 h at 37°C. Then, the cells were treated with binase and/or IFNα2b for 48-72 h followed by incubation with WST-1 reagent for 60 min at 37°C. The absorbance of samples was measured in a multiscan FC microplate reader (Thermo Fisher Scientific) at 450 nm. A mixture of cell-free medium with the WST-1 reagent was used as a background control. The activity of mitochondrial dehydrogenases was calculated as the difference in absorbance between each sample and the background control. Respiratory activity of untreated cells was taken as 100%. The experiment was performed in triplicate and reported as mean± SD.

Mitochondrial activity as a measure of cytotoxicity was determined in KYSE510 esophageal carcinoma cells with the WST-1 test kit (Roche Applied Science, Mannheim, Germany). 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium is reduced to a water-soluble formazan salt by mitochondrial enzymes of the cells. The formazan salt can be detected photometrical. 1,300 KYSE510 cells/well were seeded in 96-well plates and cultivated for 48 h. Thereafter, cells were incubated with AOH and 4-OH-AOH or the positive control Triton X-100 for 24 h in serum-containing medium. The assay was performed according to the manufacturer’s protocol, and absorbance was measured at 450 nm with a reference wavelength of 650 nm with a plate reader (Victor³ V, Perkin Elmer, Waltham, MA, USA). Cell viability was specified as mitochondrial activity and calculated as treated cells to control cells × 100 (% T/C).

In order to determine the effects on cell toxicity and viability, the commercially available WST-1 test kit was used (Roche Applied Science, Mannheim, Germany). The test principle is based on the ability of cells to reduce the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate to a water soluble formazan salt, which can be measured in a plate reader at λ = 450 nm. Cells were seeded into a 96-well plate and allowed to grow for 48 h (cell confluence of around 70%). Thereafter, cells were incubated with the respective SiO₂ NPs prepared according to the SOP for 45 min or 24 h. Triton X-100 was included in the test system as a positive control. Then, cells were washed twice with 100 µL PBS and incubated with 110 µL of a 1:10 WST-1 reagent/medium dilution (v/v). Subsequently, the plates were measured including a reference wavelength of λ = 650 nm. Each SiO₂ NPs preparation was tested three times with cells from at least three different passages and every concentration was measured six times resulting in eighteen replicates for each experimental point. The effect on the mitochondrial activity was expressed as percentage of viability (treated-over-control ratio × 100%).