Cytotoxicity Assay (MTT)

The cell metabolism of HaCaT cells was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay uses the ability of the mitochondrial succinate dehydrogenase enzyme to convert the yellow salt of MTT to the purple formazan crystalline which is insoluble in water. After the addition of dimethyl sulphoxide (DMSO) as solvent, formazan is solved, and the produced color is measured at the specified wavelength by using a microplate reader. Briefly, 96-well plates were seeded with 3X10⁵/mL HaCaT cells, and, after 48 h of incubation, monolayers were treated with different NE concentrations ranging from 5.0 to 0.005 mg/mL of surfactant (Tw80). After 24h, the medium was removed, the cells were washed with phosphate-buffered saline (PBS) and, subsequently, incubated with 0.5 mg/mL MTT (Sigma-Aldrich, St. Louis, MO, USA) for additional 4 h. Finally, the medium was removed, the cells were washed with PBS for three times, and then, 100 μL of DMSO (Sigma-Aldrich, St. Louis, MO, USA) was added to dissolve the formazan crystals. Optical density (OD) at 570 nm was determined with a spectrophotometer/fluorimeter microplate reader (PerkinElmer, Hopkinton, MA), and the cell viability for each concentration was estimated through comparison with untreated cells. HaCaT cells cultured in DMEM without the addition of compounds were used as a control. Ten percent of DMSO was used as positive control.