Transwell clear permeable filter supports

Cultures were established as previously described^{41 (link),42 (link)}. Briefly, C57BL/6 mice were sacrificed at 2–4 months of age, trachea were excised, opened longitudinally to expose the lumen, and placed in 1.5 mg/mL Pronase E in DMEM/F12 medium (Life Technologies) at 4 °C overnight. Tracheal epithelial cells were dislodged by gentle agitation and collected in DMEM/F12 with 10% FBS. After centrifugation, cells were treated with 0.5 mg/mL DNase I for 5 min on ice and centrifuged at 4 °C for 10 min at 400 g. Cells were resuspended in DMEM/F12 with 10% FBS and plated in a tissue culture dish for 5 h at 37 °C with 5% CO₂ to adhere contaminating fibroblasts. Non-adhered cells were then collected, concentrated by centrifugation, resuspended in an appropriate volume of mTEC-Plus medium (described in ref. ^{42 (link)}), and seeded onto Transwell-Clear permeable filter supports (Corning). Air-liquid interface (ALI) was established 2 days after cells reached confluence by feeding mTEC-Serum-Free medium^{42 (link)} only in the lower chamber. Cells were cultured at 37 °C with 5% CO₂, and media replaced every 2 days, and fixed on the indicated days. All chemicals were obtained from Sigma Aldrich unless otherwise indicated. Media were supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL Fungizone (all obtained from Life Technologies).