Keratinocyte Culture and Stimulation Protocol

Normal human epidermal keratinocytes (NHEKs) were purchased from MilliporeSigma and cultured for 3–4 passages using the EpiGRO™ Human Epidermal Keratinocyte Complete Media Kit (MilliporeSigma, Burlington, MA, USA). Human skin-equivalent models (HSEMs) were purchased from MetTek. HSEMs were cultivated according to the manufacturer’s instructions. NHEKs were grown in a humidified atmosphere containing 5% CO₂ and 95% air atmosphere. Cells were seeded at a density of 2 × 10⁴ cells/well in a flat-bottomed 96-well microculture plate and were cultured until they reached 90% confluence, then the media was changed to fresh medium without hydrocortisone. After further cultivation for 24 h, keratinocytes were stimulated using previously described methods with CCs [52 (link),53 (link)] that included: TNF-α (20 ng/mL, R&D system), IL-4 (100 ng/mL, R&D system), IL-13 (100 ng/mL, R&D system) and flagellin (100 ng/mL; Peprotech, Inc., Seoul, Korea). Kushenol F pretreatment was performed for 1 h before keratinocyte activation.