Costar 12 mm 0.4 μm transwells

BECs were used at passage less than or equal to 3. Cells were seeded onto Corning Costar 12 mm 0.4 μm Transwells® (Corning® Life Sciences) coated with type I collagen at a concentration of 100,000 cells/transwell. Cells were then kept in submerged culture using BEGM in both the apical and well chambers until confluent. Once confluent, cells were then changed to Pneumacult^tm ALI Media in the lower well chamber only and the remaining apical media was aspirated. ALI media in the basolateral compartment was changed every other day and cells were differentiated at an ALI for 21 days prior to initiation of cell culture experiments.

BECs were used for these studies at each passage corresponding number. Once cells were ~ 70-90% confluence in flasks, they were trypsinized with 1 mL of 0.025% Trypsin-EDTA and then seeded onto collagen I pre-coated (Collagen Solution, STEMCELL™ Technologies) Corning Costar 12 mm 0.4 μm Transwells® (Corning® Life Sciences) at a concentration of 100,000 cells per transwell. Cells were then kept in submerged culture using BEGM in both the apical and basolateral well chambers for 7 days or until confluent. Once confluent, cells were then changed to Pneumacult™ ALI Medium (StemCell™ Technologies) in the lower basolateral chamber only and the remaining apical media was aspirated. ALI media in the basolateral compartment was changed every other day and cells were differentiated at an ALI for 21 days.