Ursolic Acid Cytotoxicity Assay

HaCaT cells were seeded on 96-well plates (5000 cells/well) a day prior to the experiment. Cells were incubated with various concentrations of ursolic acid formulations in the presence of 1 ng/mL M5 cytokine mix diluted in DMEM medium supplemented with 1% FBS. After 72 h, the medium was removed, and cells were washed once with PBS and incubated with 200 µL/well of 0.5 mg/mL Thiazolyl Blue Tetrazolium Bromide (Merck, Poznań, Poland) in DMEM for up to 20 min at 37 °C/5% CO₂. The medium was removed again, and formazan crystals were dissolved in 120 µL of isopropanol acidified with 5 mM hydrochloric acid (Avantor Performance Materials, Gliwice, Poland). Quantities of 90 µL of samples from each well were transferred to a new, transparent 96-well plate. Absorbance of samples was measured using Spectra Max Gemini EM (Molecular Devices, San Jose, CA, USA) at 570 nm. Results were calculated as a percentage of the untreated control (cells in DMEM medium).