P2300 ussing chambers

Short-circuit currents were measured in CF ALI cultures generated from LV-dt/EGFP-RmiRT and LV-dt/EGFP-miRT transduced basal cells following differentiation for at least 3 weeks. Transwells were placed under VCC MC8 voltage clamps and P2300 Ussing chambers (Physiologic Instruments, San Diego, CA, USA) with low chloride buffer in the apical chamber and high chloride buffer in the basal chamber, as previously described [12 (link),13 (link)]. The change in current was assessed after the sequential addition of the following antagonists and agonists: 100 μM amiloride (ENaC inhibitor), 100 μM 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) (a general chloride channel blocker that does not affect CFTR), 100 μM 3-Isobutyl-1-methylxanthine (IBMX) and 10 μM forskolin (to increase intracellular cAMP levels which activate CFTR), and 50 μM GlyH101 (a CFTR channel blocker).

CFTR-dependent ion transport electrophysiology was measured in HBEC cultures by I_sc using modified P2300 Ussing chambers (Physiologic Instruments, San Diego, CA) as previously described (37 (link), 38 (link)). Increasing concentrations of AZD5634, benzamil, or vehicle, followed by the addition of benzamil (10 μM), benzamil plus low chloride (to establish a chloride gradient), forskolin (20 μM), and CFTRinh-172 (10 μM) were added sequentially. All chambers were maintained at 37°C and agonist stimulation was initiated within 15 min of placement in the chambers.

CFTR function was assessed in murine nasal epithelium by nasal potential difference (NPD) measurements and in murine trachea by analysis of short-circuit current (Isc) according to previously published methods [9 (link)]. Briefly, for NPD assessment, mice were anesthetized and sequentially perfused with Ringer’s solution (baseline); Ringer’s plus amiloride (100 μm) and a chloride-free solution containing K₂HPO₄ (2.4 mM), KH₂PO₄ (0.4 mM), Na Gluconate (115 mM), NaHCO₃ (25 mM), and Ca₂ Gluconate (1.24 mM) with forskolin (20 μM); and roflumilast (30 nM). CFTR-dependent chloride transport was measured as the change in potential difference following perfusion with chloride-free ringers followed by forskolin or by CFTR-specific inhibitor cocktail containing 10 μM each of GlyH101 and CFTR_inh-172.
Mice were euthanized and trachea were harvested and tested as full-thickness tissue. Isc was measured under voltage clamp conditions using P2300 Ussing chambers and MC8 Voltage Clamps (Physiologic Instruments, San Diego, CA). Mounted tissues were bathed on both sides with identical Ringers solutions gassed with 95% O₂:5% CO₂ and then sequentially treated apically with amiloride (100 μM), roflumilast (30 nM), ATP (100 μM), and bumetanide (10 μM; added only to the serosal solution at the end of experiments to block chloride ion-dependent Isc).