Clear polyester membrane

Isolation and culture of primary mTECs were performed as previously described18 (link). In brief, cells isolated by enzymatic treatment were seeded onto 0.4 μm pore size clear polyester membrane (Corning) coated with a collagen solution. Cells were grown in submersion until confluent, and then exposed to air to establish an ALI. Alternatively, cells were kept in submersion or grown at an ALI in hypoxic conditions (0.5% O₂) in a Ruskinn's InvivO2 hypoxia workstation. Notch pharmacological inhibition was achieved by the treatment with DAPT (40 μM, Sigma) starting at the onset of ALI. AhR activation was achieved by treating cells with the agonists FICZ (Enzo, 250 nM) or 3MC (Sigma, 1 μM) starting from day 2 before ALI.

Isolation and culture of primary mouse airway epithelial cell culture (AEC) were performed as previously described (Crotta et al, 2013): in brief, cells were isolated from C57BL/6 mouse trachea by enzymatic treatment and seeded onto a 0.4‐μm pore size clear polyester membrane (Corning) coated with a collagen solution. At confluence, medium was removed from the upper chamber to establish an air–liquid interface (ALI). Fully differentiated, 7‐ to 10‐day‐old post‐ALI cultures were routinely used for experiments. For analysis of cytokine secretion, AEC cultures were stimulated with IFNα (0.725 ng/ml), IFNλ (1.3 ng/ml) or LPS (1 μg/ml, Lonza) or medium control for 24 h. Supernatants were then collected and stored at −70°C until samples were analysed.