CFTR Function Assessed in Airway Epithelial Cells

CFBE41o-cells expressing WT or F508del CFTR were seeded onto permeable supports (Costar) after coating with fibronectin [24 (link)]. Cells were grown to confluence and transferred to an air—liquid interface, after which they were mounted in modified Ussing chambers. Monolayers were initially bathed on both sides with identical Ringers solution containing (in mM): 115 NaCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.24 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, 10 D-glucose (pH 7.4) and vigorously stirred and gassed with 95%O₂: 5% CO₂ at 37°C. Short-circuit current (Isc) was obtained using an epithelial voltage clamp. The mucosal bathing solution was changed to a low Cl^- solution containing (in mM): 1.2 NaCl, 115 Na gluconate, plus 100 μM amiloride followed by addition of agonists (20 μM forskolin, 50 μM genistein, and/or 10 μM ivacaftor) to the mucosal surface. CF inhibitor 172 (10 μM; Sigma-Aldrich, St. Louis, MO) was added to the bathing solution at the conclusion of each experiment to block CFTR-dependent Isc.