Culturing and Treating Normal Epidermal Keratinocytes

Normal human epidermal keratinocytes (NHEKs) obtained from Lonza (Basel, Switzerland) were grown in culture dishes at 37 °C in 5% CO₂^{35 (link)}. The NHEKs were cultured in serum‐free keratinocyte growth medium supplemented with bovine pituitary extract, recombinant epidermal growth factor, insulin, hydrocortisone, transferrin, and epinephrine (all from Lonza). Culture medium was replaced every 2 days. Near confluence (70%–90%), cells were disaggregated with 0.25 mg/mL trypsin/0.01% ethylenediaminetetraacetic acid (Lonza) and subcultured. Second‐ to fourth‐passage NHEKs were used in all experiments. The cells (1 × 10⁵) were seeded in 24‐well culture plates, allowed to attach for 24 h, and then subsequently treated with or without IL‐17 or TNF-α (PeproTech, Rocky Hill, NJ, USA) for 24 h.