Ussing Chamber Protocol for Transepithelial Resistance

Inserts were mounted in modified Ussing chambers (P2300; Physiologic Instruments, San Diego, CA) and continuously short circuited with an automatic voltage clamp (VCC MC8; Physiologic Instruments) as described previously(60 (link)-62 (link)). The apical and basolateral chambers each contained 4 ml of Ringer solution (120 mM NaCl, 25 mM NaHCO₃, 3.3 mM KH₂PO₄, 0.8 mM K₂HPO₄, 1.2 mM MgCl₂, 1.2 mM CaCl₂, and 10 mM glucose). Chambers were constantly gassed with a mixture of 95% O₂, 5% CO₂ at 37°C, which maintained the pH at 7.4 and established a circulating perfusion bath within the Ussing chamber. Simultaneous transepithelial resistance was recorded by applying a 2-mV pulse per minute via an automated pulse generator. Recordings were digitized and analyzed using PowerLab (AD Instruments, Colorado Springs, CO). To screen for aldosterone-regulated miRs from mCCD-cl1 cells in culture, equivalent open circuit currents were obtained from cells using chopstick electrodes and an Epithelial Volt/Ohm Meter (EVOM) (World Precision Instruments, Sarasota, FL). Currents were calculated from cells stimulated with or without 50nM aldosterone for 24 hrs using Ohm’s Law from the EVOM-measured open circuit voltages and transepithelial resistance.