Complete airway epithelial cell growth medium

HNECs were seeded into removable 8 well silicone cultivation chambers (ibidi, Planegg, Germany) and grown for 5 days in complete Airway Epithelial Cell Growth Medium (PromoCell) at 37 °C, 5%CO₂. At 80–100% confluence cells were fixed with 4% paraformaldehyde, rinsed, and permeabilized with PBS plus 0.1% Triton X and 0.02% SDS. Subsequently, cells were blocked with 10% goat serum, incubated with either rabbit anti-wide spectrum Cytokeratin (1:200, abcam), mouse anti-Mucin 5AC (1:100, abcam), mouse anti-acetylated alpha Tubulin (1:200, abcam), mouse anti-Cytokeratin 14 (Sigma Aldrich) or rabbit anti-Claudin-1 (1:50, Invitrogen), rabbit anti-ZO-1 (1:50, Invitrogen), mouse anti-Occludin (1:50, Invitrogen) and then incubated with secondary antibodies: goat anti-mouse AF488 (1:2000, Invitrogen), donkey anti-rabbit AF488 (1:2000, Invitrogen) or goat anti-rabbit PE (1:100, Santa Cruz, Dallas, US). Images were recorded on a DMi8 microscope using LAS X Life Science microscope software (Leica, Wetzlar, Germany).

HNECs were seeded into 24 or 12 well plates, at densities of 5 × 10⁴ cells/ml or 1 × 10⁵ cells/ml, respectively, in complete Airway Epithelial Cell Growth Medium (PromoCell) and grown for 5 days at 37 °C, 5% CO₂. At 80% confluence, cultures were changed to Airway Epithelial Cell Growth Medium without hydrocortisone and treated with either flagellin (100 ng/ml), lipopolysaccharide (LPS; 100 ng/ml), Pam3Cys (1 μg/ml), PolyIC (10 μg/ml), CPG (5 μg/ml), nigericin (10 ng/ml), or APEs (1.0  μg total protein/ml). Unstimulated HNECs served as control. After 24 h IL-1β, CCL-20, CCL-5, CCL-22, IL-33 (R&D Systems, Minneapolis, US) IL-1α, CCL-2, IP-10, GM-CSF, IL-8 (BD Pharmingen, San Jose, US), IL-18 (eBioscience, San Diego, US) and HBD-2 (Peprotech, Hamburg, Germany) were measured in cell culture supernatants by ELISA.