Easymount ussing chambers

Cultures were treated with 3 µM elexacaftor (E) and 18 µM tezacaftor (T) or with 0.07% dimethyl sulfoxide (DMSO) (vehicle control) for 24 before Ussing chamber analysis. ivacaftor was added acutely during transepithelial short-circuit current (I_sc) measurements to ET pre-treated cultures, to provide conditions that maximally enhance CFTR function. CFTR modulators were obtained from Selleck Chemicals (Planegg, Germany), DMSO was obtained from Sigma-Aldrich (St Louis, MO, USA). I_sc was measured by EasyMount Ussing chambers (Physiologic Instruments, San Diego, CA, USA) as previously described, in presence of a chloride gradient (basolateral 145 mM vs. apical 5 mM) [22 (link),42 (link)]. After 10–20 min equilibration, amiloride (100 µM) was added to inhibit sodium absorption via the epithelial sodium channel (ENaC). To assess CFTR-mediated chloride secretion, forskolin (Fsk, 10 µM) and 3-isobutyl-1-methylxanthin (IBMX, 100 µM) were applied together, then ivacaftor (2.5 µM) was added, followed by CFTR inhibitor-172 (CFTRinh-172, 20 µM, TargetMol chemicals, Boston, MA, USA). All Ussing chamber chemicals and reagents, apart from CFTRinh-172 and ivacaftor, were obtained from Sigma-Aldrich (St Louis, MO, USA) at the highest level of available purity. Bioelectric responses were quantified by LabChart8 (AF Instruments, Oxfordshire, UK).

Human and animal airway tissue preparations and cultured cells on filters were mounted in EasyMount Ussing chambers (Physiologic Instruments, CA) with exposed surface areas of 0.07~1.13 cm² depending on the preparation, and were bathed in KRB buffer at 37 °C with a continuous 95% O₂ and 5% CO₂ gas supply, except for HCO₃⁻-free buffer solutions which were gassed with pure O₂. Transepithelial short-circuit current (Isc) was measured using a VCC-600 voltage clamp (Physiologic Instruments, CA) and Isc data were obtained and displayed with PowerLab Chart4 software (ADInstruments, CA). Total tissue conductance was calculated by applying Ohm’s law to the Isc deflection resulting from 1 mV pulse across the tissues/cell cultures every 20 sec during the experiment (Supplementary Table S.1).

Transepithelial measurements were performed on Caco-2 cells grown as monolayers for 20 days. Inserts were mounted into Easy Mount Ussing chambers (Physiologic Instruments Inc., Reno, NV, USA).
Apical and basolateral hemi-chambers were filled with 5 mL of Ringer’s solutions containing (in mM): 115 NaCl, 25 NaHCO₃, 0.4 KH₂PO₄, 2.4 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, and 10 glucose (pH 7.4, 300 mOsm). The solution was gassed with a 95% O₂ and 5% CO₂ mixture and constantly recirculated by gas bubble lifting.
The short circuit current (Isc) and the transepithelial resistance (Rt) were measured as previously described [31 (link)].