Airway Epithelial Electrophysiology Assay

HBECs were exposed to pooled 15 μL BALF per culture for 6 h vs. PBS (control). After this time, perfluorocarbon (Thermo Fisher Scientific, Waltham, MA, USA, cat no. 394890500) was added to prevent evaporation and membrane potentials were recorded using a KCl-filled glass microelectrode that was positioned in the ASL with a micromanipulator and a KCl/agar macroelectrode that was positioned in the serosal media. Transepithelial voltages were then recorded using an FD223 electrometer (World Precision Instruments, Sarasota, FL, USA) as described [23 (link),26 (link)]. Changes in voltages caused by CFTR-mediated transepithelial Cl⁻ secretion and ENaC-mediated transepithelial Na absorption were determined by calculating bumetanide and amiloride-inhibitable voltages, respectively. We then measured resistance using an Epithelial Volt Ohm Meter (EVOM2, World Precision Instruments, Sarasota, FL, USA) as described [48 (link)]. Equivalent open circuit currents (I_eqs) were then calculated according to Ohm’s law.