Vcc 600 transepithelial clamp

Chambers for mounting either transwell cell cultures or mouse tissue biopsies were obtained from Physiologic Instruments (model P2300, San Diego, CA, USA). Chamber solution was buffered by bubbling with identical Ringer solution on both sides and were maintained at 37°C, vigorously stirred, and gassed with 95%O2/5% CO2. Cells or tissues were short circuited using Ag/AgCl agar electrodes. A basolateral-to-apical chloride gradient was established by replacing NaCl with Na-gluconate in the apical (luminal) compartment to create a driving force for CFTR-dependent Cl⁻ secretion. To measure stimulated Isc, the changed sodium gluconate solution, after stabilization, was supplied with 100µM amiloride. Agonists (forskolin) were added to the bathing solutions as indicated (for a minimum 5 min of observation under each condition) to activate CFTR channels present at the apical surface of the epithelium (either cell surface or lumen side of the tissue) and CFTR_Inh-172 (10µM) was added to the mucosal bathing solution to block CFTR-dependent Isc. Short-circuit current (expressed as Isc (μA/cm2)) and resistance were acquired or calculated using the VCC-600 transepithelial clamp from Physiologic Instruments and the Acquire&Analyze2∙3 software for data acquisition (Physiologic Instruments), as previously described [12 (link),71 (link)–74 (link)].

Chambers for mounting mouse tissue biopsies were obtained from Physiologic Instruments (model P2300, San Diego, CA, USA). Chamber solution was buffered by bubbling with identical Ringer solution on both sides and were maintained at 37 °C, vigorously stirred, and gassed with 95%O₂/5%CO₂. Tissues were short circuited using Ag/AgCl agar electrodes. A basolateral-to-apical chloride gradient was established by replacing NaCl with Na-gluconate in the apical (luminal) compartment to create a driving force for CFTR-dependent Cl⁻ secretion. To measure stimulated Isc, the changed sodium gluconate solution, after stabilization, was supplied with 100 µM amiloride. Agonists (forskolin) were added to the bathing solutions as indicated (for a minimum 5 min of observation under each condition) to activate CFTR channels present at the apical surface of the epithelium (either cell surface or lumen side of the tissue) and CFTR_Inh-172 (10 µM) was added to the mucosal bathing solution to block CFTR-dependent Isc. Short-circuit current (expressed as Isc (μA/cm²)) and resistance were acquired or calculated using the VCC-600 transepithelial clamp from Physiologic Instruments and the Acquire &Analyze2∙3 software for data acquisition (Physiologic Instruments), as previously described^{18 (link),54 (link)}.