Pneumacult ali maintenance supplement

All cells were grown at 37 °C in a humidified atmosphere with 5% (v/v) CO₂. Culture conditions for CFBE and HT₂₉ cells have been described earlier [25 (link)]. In brief, airway epithelial cells were grown in DMEM/Ham’s F-12 with L-Glutamine medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine 200mM and 1% (v/v) HEPES 1M (all from Capricorn Scientific, Ebsdorfergrund, Germany). CFBE parental cells were grown in MEM with Earle’s Salts with L-Glutamine medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS. The airway epithelial cell line H441 was grown in RPMI and DMEM media. The immortalized human airway basal cell line BCi-NS1 (kindly provided by Prof. Ron Crystal, Weill Cornell Medical College, New York City, NY, USA) was maintained in Bronchial Epithelial Growth Media (Lonza). Cells were differentiated by growing on permeable supports (Snapwell, Corning, NY, USA) in an air-liquid interface (ALI) for up to 30 days in PnemaCult-ALI medium supplemented with PneumaCult-ALI 10X Supplement, PneumaCult-ALI Maintenance Supplement, hydrocortisone and heparin (all from StemCell Technologies, Vancouver, BC, Canada), and 1% penicilin-steptomycin (10 000 U/mL; Gibco, ThermoFisherScientific, Waltham, MA, USA).

mesalazine solution was prepared by dissolving powdered mesalazine (>99.9% purity) (Sigma Aldrich, St. Louis, MO, USA) in sterilised Mili Q water and adjusted to pH to 7 using 1 mM sodium hydroxide (NaOH) solution. The mesalazine solution was then diluted into either PneumaCult™-Ex Plus Basal Medium (STEMCELL Technologies, Tullamarine, VIC, Australia), PneumaCult™-Ex Plus 50X Supplement (STEMCELL Technologies, Tullamarine, VIC, Australia), and penicillin-streptomycin (Thermo Scientific, Walthman, MA, USA) (designated as Ex Plus complete media)] or PneumaCult™-ALI Basal Medium (STEMCELL Technologies, Tullamarine, VIC, Australia); PneumaCult™-ALI 10X Supplement; penicillin-streptomycin/amphotericin B (Thermo Scientific, Waltham, MA, USA); And PneumaCult™-ALI Maintenance Supplement (STEMCELL, Vancouver, BC, Canada) (ALI complete media)] to achieve the final concentrations of 0.5 mM, 1 mM, 10 mM, 20 mM and 50 mM. The mesalazine solution was covered with foil paper in all experiments unless specified.