Pneumacult ali differentiation medium

HBE cells were cultured on T75cm² flasks (Costar, Corning Inc., Corning, NY) in Bronchial Epithelial Growth Medium (BEGM, Lonza, Walkersville, MD) supplemented with 5% FBS for 72 h (Life Technologies, Burlington, Ontario, Canada). Cells were then fed every 48 h with BEGM without FBS. At 90% confluence, cells were lifted and seeded at 2.0 × 10⁵ cells per insert onto 1.12cm², 0.4 μm pore transwell inserts (Costar) coated with bovine collagen Type I/III (Advanced BioMatrix, San Diego, CA), and cultured in BEGM for 48 h. BEGM was then removed and HBE were cultured using only basolateral PneumaCult-ALI differentiation medium containing 100X supplement, hydrocortisone, and heparin (Stemcell Technologies, Vancouver, BC, Canada), as well as fluconazole (Sigma-Aldrich, Oakville, Ontario, Canada) and penicillin/streptomycin (Life Technologies). Cells were fed basolaterally every 48 h. Beginning 14 days after seeding, cells were washed apically once per week with PBS to remove excess mucus. Cultures were used for experiments at 5 weeks after transwell seeding.

HEPES (4-(2-hydroxyethyl)–1-piperazineethanesulfonic acid), PEI 25 kDa, heparin sodium salt, Paraformaldehyde solution, FluorSave™ Reagent, Eagle’s Minimum Essential Medium (EMEM), RPMI-1640 Medium, fetal bovine serum (FBS), Penicillin-Streptomycin solution, Dulbecco’s Phosphate Buffered Saline (PBS), trypsin-EDTA solution, 200 mM L-glutamine solution, Paraformaldehyde, Tween20, agarose and Alcian Blue solution (1% in 3% acetic acid pH 2.5) were purchased from Sigma-Aldrich (Darmstadt, Germany). Lipofectamine 2000, SYBR gold dye, AF488-anti-rabbit secondary antibody, rhodamine phalloidin, 4′,6–diamidino–2-phenylindole dihydrochloride (DAPI), Alexa Fluor™ 647 NHS ester and Alexa Fluor™ 488 NHS ester were obtained from Life technologies (Carlsbad, California, USA). Transwell® polyester membrane cell culture inserts (0.4 μm pore size) were purchased from Corning (New York, USA). PneumaCult ALI differentiation medium, hydrocortisone and heparin were purchased from Stemcell Technologies (Vancouver, Canada). Alveofact was purchased from Lyomark Pharma (Oberhaching, Germany). ROTI®GelStain Red and bovine serum albumin were purchased from Carl Roth GmbH (Karlsruhe, Germany). Dicer substrate double-stranded siRNA (DsiRNA) targeting human GAPDH, non-specific DsiRNA and amine-modified siRNA were purchased from integrated DNA Technologies (Leuven, Belgium).