2.4. KeratinoSensTM Assay Test Methods

A transgenic human keratinocyte cell line with a stable insertion of the Luciferase reporter gene under the control of the Antioxidant Response Element (ARE)-element KeratinoSens^TM cells was provided by Givaudan Suisse SA (Vernier, Switzerland). The cells were cultured in DMEM media supplemented with 10% FBS, 0.5 mg/mL Geneticin (Sigma-Aldrich). The cells were sub-cultured every 2–4 days at 80–90% confluence for a maximum of 25 passages. The culture medium was replaced to a fresh medium and incubated in a humidified atmosphere condition of 5% CO₂ at 37 °C. Stabilized KeratinoSens^TM cells were seeded into a 96-well cell culture plate at a density of 1 × 10⁴ cells/well and incubated overnight. The prepared cells were washed once with pre-warmed pH 7.4 Dulbecco’s phosphate buffered saline (DPBS), followed by the addition of dispersed silica NPs suspension (0.98–2000 μM), and the culture plates were then incubated for 48 h. Positive control, cinnamic aldehyde (CASRN. 14371-10-9, Sigma-Aldrich), was tested in parallel (concentration: 4–64 μM). The viability of KeratinoSens^TM cells was measured using the thiazolyl blue tetrazolium bromide (3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay reduction test (Promega, Madison, WI, USA). To exclude the colorimetric interference from silica NP present in the cells, the supernatant was transferred into clear 96-well plates, and the absorbance was measured at 570 nm with a multi-microplate reader (Synergy HTX, BioTek, Winooski, VT, USA). The cell viability (%) was calculated based on the optical density (OD) of the vehicle control and blank control. Then, to measure the luciferase activity of silica NP, we used the One-Glo^TM Luciferase assay kit (Promega). The luciferase assay was conducted under the same conditions as the MTT assay. The luminescence intensity of each sample was measured using a multi-microplate reader. Levels of luciferase intensity were calculated based on the luminescence values of the vehicle control and blank control.