Ussing chamber studies of primary nasal epithelial cells

Day 14 ALI nasal epithelial cells in 6.5-mm transwells were mounted in circulating Ussing chambers (Physiological Instruments, San Diego, CA, USA) and measurements to assess CFTR function were performed as previously described [26]. The perfusion bath (126 mM NaCl, 24 mM NaHCO₃, 2.13 mM K₂HPO₄, 0.38 mM KH₂PO₄, 1 mM MgSO₄ 1 mM CaCl₂ and 10 mM glucose) was maintained at a pH of 7.4, a temperature of 37°C and continuously gassed with a 5% CO₂/95% O₂ mixture. Measurements were performed in symmetrical chloride concentrations, in open-circuit mode, and are presented as transepithelial current (I_eq; μA·cm⁻²). The transepithelial resistance was 250±122 Ω·cm⁻² for healthy control nasal cells and 330±130 Ω·cm⁻² for CF nasal cells. Before the experiments, cells were treated with 0.1% DMSO, 3 µM VX-809+1 µM VX-770, 3 µM VX-661+3 µM R-VX-445+1 µM VX-770 or 3 µM VX-661+3 µM S-VX-445+1 µM VX-770 for 48 h. During the experiment 1 µM VX-770 was also added acutely. CFTR function was determined in the presence of amiloride (30 µM; Spectrum Chemical, Gardena, CA, USA) and following cAMP activation with FSK (I_eqFSK, 0.1 or 10 µM for wild-type (WT) or 10 µM for F508del-, M1101K-, G85E- and N1303K-CFTR; Sigma-Aldrich, St Louis, MO, USA). CFTR activity was confirmed as I_eq difference following CFTR inhibition with CFTRinh-172 (I_eqCFTRinh-172, 10 µM) [20, 27]. The maximal response I_eqFSK was calculated as the difference between the steady baseline of the measured I_eq following amiloride inhibition and the lowest measured peak I_eq following addition of ivacaftor/elexacaftor plus FSK.