Transferrin

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.

The non‐transformed porcine intestinal epithelial cell line IPEC‐J2, originally isolated from jejunal epithelia of a neonatal unsuckled piglet (Schierack et al., 2006 (link)), was a kind gift of Dr. Jody Gookin, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, NC, USA. IPEC‐J2 cells were grown and maintained in complete medium, which consisted of a 1:1 mixture of Dulbecco's modified eagle's medium and Ham's F‐12 Nutrient Mixture (DMEM/F12) (plain medium) supplemented with 5% foetal bovine serum (FBS), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium, 5 ng/ml epidermal growth factor and 1% penicillin–streptomycin (all from Lonza Group Ltd, Belgium). Cells were grown at 37°C in a humidified atmosphere of 5% CO₂.
IPEC‐J2 cells were seeded onto six‐well plates (Corning Inc., Corning, NY, USA), coated with 8 μg/cm² rat tail collagen type I (Sigma–Aldrich, Steinheim, Germany), at a density of 10⁶ cells/ml; the volume of complete medium was 2.5 ml. Cells could adhere for 24 h before being washed and re‐fed every other day.

The IPEC-J2 cell line was provided by Juan José Garrido from the Department of Genetics and Animal Breeding, University of Cordoba, Spain. IPEC-J2 cells were grown and maintained in DMEM/F-12 (Sigma-Aldrich, St. Louis, MO, USA), supplemented with fetal bovine serum (5% FBS; Lonza, Switzerland), glutamine (2 mM; Biosera), epidermal growth factor (5 ng/mL; BD Biosciences, San José, CA, USA), transferrin (10 μg/mL), insulin (10 μg/mL), selenium (10 ng/mL) (Lonza), and gentamicin (50 μg/mL; Sigma-Aldrich). Cells were incubated at 37 °C in a fully humidified atmosphere with 5% CO_2, until confluence. Cells were seeded in 6-well culture plates (TPP, Switzerland) (1.5 × 10⁵ cells per well) 72 h before the experiment, and cultured in DMEM/F-12 medium as mentioned above, but supplemented with hydrocortisone (0.28 μM; Sigma-Aldrich) and ascorbic acid (5 μg/mL; Sigma-Aldrich) to avoid preliminary cell-activation. At 24 h prior to the experiment, the cell medium was changed to DMEM/F-12 without supplementation (without FBS and gentamicin). The cultures were regularly controlled for the absence of mycoplasma contamination [13 (link)].