Bronchial ASL pH measured in vitro using fluorescence imaging

Air–liquid interface cultures contained columnar cells with beating cilia and goblet cells with mucin granules. Mucus accumulated on the apical surface and was removed before most experiments by rinsing with PBS containing 5 mM N-acetylcysteine. Bronchial epithelial cultures were mounted in a closed chamber during CO₂ oscillations on the stage of an inverted fluorescence microscope (IX81; Olympus) equipped with an imaging system (Photon Technology International) as described (Kim et al., 2019). Cells were rinsed with artificial ASL (mM: 130 NaCl, 10 NaHCO₃, 5 KCl, 1 CaCl₂, 1 MgSO₄, 2.8 NaHepes, 2.2 Hepes acid, and 10 glucose) and covered with 30 µl artificial ASL for at least 30 min before measurements. The ratiometric fluorescent pH indicator BCECF conjugated to dextran (Molecular Probes; Thermo Fisher Scientific) was added to artificial ASL using the vehicle FC-72, a perfluorocarbon that evaporated rapidly from the surface. To mimic tidal breathing, cells were exposed to alternating flows of 0.035% and 5% CO₂ using a custom-designed gas-mixing pump equipped with a humidification system and interfaced to a computer (Okolab). CO₂ levels at the gas inlet and outlet ports of the perfusion chamber were measured using an infrared CO₂ analyzer (CD-3A; Applied Biosystem). In some experiments, purified carbonic anhydrase type 2 (1 unit; Sigma-Aldrich) or the carbonic anhydrase inhibitor acetazolamide (100 µM) or topiramate (200 µM) was added to the artificial ASL. The effects of mucins on pH oscillations were studied by adding porcine gastric mucins (20–80 mg/ml; Sigma-Aldrich) dissolved in artificial ASL.