Mc8 equipment

CFBE or HBE cells expressing WT- or ΔF508-CFTR were grown to confluence on permeable filters at an air-liquid interface and mounted in modified Ussing chambers. I_sc measurements were obtained under voltage clamp conditions using MC8 equipment and P2300 Ussing chambers (Physiologic Instruments) as previously described [51 (link),52 (link),103 (link)]. Cells were equilibrated for 5–10 min in regular Ringer solution (115 mM NaCl, 25 mM NaHCO₃, 2.4 mM KH₂PO₄, 1.24 mM K₂HPO₄, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM D-glucose, pH 7.4). In some measurements, this was followed by the exchange of low chloride Ringer (115 mM NaCl reduced to 1.2 mM NaCl and 10 mM D-glucose replaced with 115 mM Na-gluconate) to the apical surface. After addition of the sodium channel inhibitor amiloride (100 μM), the CFTR agonists Frk (10 μM) and gen (50 μM) were sequentially added, followed by inhibitor₁₇₂ (10 μM) at the conclusion of each experiment, in order to specifically inhibit CFTR activity. Changes in CFTR-mediated ion transport were calculated using the highest current value for each sample after achievement of a stable plateau for several minutes.

Tracheas were excised, sectioned into 3–4 segments, and opened longitudinally along the dorsal surface. Segments were mounted as flat sheets in modified Ussing chambers (area ∼0.031 cm²) maintained at 37°C and bubbled vigorously with 95% O₂∶5% CO₂.
I_SC measurements were performed under voltage clamp conductions using MC8 equipment and P2300 Ussing chambers (Physiologic Instruments, San Diego, CA). Tissue segments were equilibrated for 10 minutes in regular Ringer solution that contained (in mM) 120 NaCl, 25 NaHCO₃, 3.33 KH₂PO₄, 0.83 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, and 10 mannitol to establish a baseline and then tested by one of the following experimental protocols:
Changes in I_SC attributable to ion transport agonists and inhibitors were calculated following achievement of a stable plateau for several minutes. ATP-sensitive I_SC was measured as the highest current value for each sample [21] (link).