Tox 1 kit

Cell viability was assessed by measuring their ability to metabolize 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) using the TOX-1 kit (Sigma Aldrich) at 24 hr timepoint after 100 nM atRA and C3 treatment.

Stromal (n = 5) and epithelial (n = 5) cells derived from passage I were placed in a 96-well plate. After reaching 50% of confluence, the medium was replaced with fresh DMEM without phenol red supplemented with 0.1% BSA and antibiotics and antimycotic solution. Then, cells were incubated with vehicle or with IL-1α, IL-1β, or IL-6 (10 ng/mL each). After 24 h, cells proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method using TOX-1 Kit (Sigma-Aldrich, Madison, USA, #7H258), according to the manufacturer's instructions.

Immortalized human keratinocytes viability was estimated by the ability of the cells to enzymatically reduce 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide (MTT) to formazan (Tox1 kit; Sigma‐Aldrich, St. Louis, MO, USA). HaCaTs were plated at 25 000 cells per well in a 96‐well plate (100 μL per well) and grown to confluence for 24 h at 37 °C, 5% CO₂. The cells were treated with LL‐37 or CSAs in full growth medium. After 24 h of additional incubation, the cell supernatant was removed and replaced with MTT (10 μL per well). After 4 h of incubation at 37 °C, 5% CO₂, the MTT crystals were dissolved using MTT Solubilization Solution (100 μL per well). After 1 min of shaking, absorbance was read at 570 nm/corr: 690 nm (Synergy™ HT, Biotek). Percent viability was determined by normalizing absorbance to that of control cells treated only with full growth medium. Data presented as mean ± SEM for N = 4 experiments.