Keratinocyte Differentiation and Cytokine Regulation

Human keratinocytes were isolated from neonatal foreskin as previously described [35 (link)]. PHK were cultured in Keratinocyte-SFM (Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). To differentiate PHK, cells were grown in DMEM (11965092, Thermo Fisher Scientific, Waltham, MA, USA) with 1% Pen/Strep, 0.2% Amphotericin B. For cytokine experiments, the following reagents were added alone or in combination to the culture media from the time of differentiation and replaced with every 48-h media change: human IL-4 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-13 (5–50 ng/mL; R&D system, Minneapolis, MN, USA), human IL-17A (1–100 ng/mL; R&D system), JAK inhibitor I (10 μM; Calbiochem, San Diego, CA, USA), and PD98056 (10 μM; Calbiochem, San Diego, CA, USA). For the epidermal organotypic model experiment, keratinocytes were grown as previously described [36 (link)]. For all cytokine treatment studies, keratinocytes were starved of growth factors for 24 h before stimulation with the indicated cytokines.