Fluorinert 77

Primary tracheal epithelial cultures were washed with PBS, and 20 μl of PBS containing 2 mg/ml Rhodamine dextran (10 kDa; Molecular Probes) was added to the lumen to visualize the airway surface liquid layer. To avoid evaporation of the ASL, 80 μl of immiscible perfluorocarbon (Fluorinert-77, Sigma-Aldrich) was added to the airway surface following the addition of the labeling dye. Images of the Rhodamine-labeled airway surface liquid were acquired by confocal microscopy (Leica TCS SP8, Leica Microsystems, Mannheim, Germany). The height of the airway surface liquid was measured by averaging the heights obtained from xz scans of sixteen predetermined positions on the culture as previously described [22 (link)]. Airway surface liquid height was measured 5 min following the addition of the Rhodamine dextran and at designated time points over a period of 24 h in primary tracheal epithelial cultures from βENaC-Tg mice and WT littermates.

Four to 6-week-old mice were induced with doxycycline for 2 weeks. Tracheae from ten mice per experimental group were freshly excised as described above, pooled and epithelial cells were isolated and cultured on membranes (T-Col, Costar) under air–liquid interface conditions as previously described^{78 (link)}. After reaching confluence (14 days) primary tracheal epithelial cultures were washed with PBS and 20 µl of PBS containing 2 mg/ml rhodamine dextran (10 kDa; Molecular Probes) was added to the apical surface to visualize the ASL layer. Adding this volume of PBS results in an initial ASL height of 25–30 µm. Totally, 80 µl of immiscible perfluorocarbon (Fluorinert-77, Sigma-Aldrich) was added to the epithelial surface following the addition of the labeling dye as described previously^{79 (link)}. Images of the Rhodamine-labeled ASL were acquired by confocal microscopy (Leica TCS SP8, Leica Microsystems) using the appropriate settings for rhodamine (excitation with 561 nm laser/emission detection at 600–650 nm). The height of the ASL was measured by averaging the heights obtained from xz scans of 16 predetermined positions on the culture. ASL height was measured at 5 min, 2 h, 4 h, 8 h, and 24 h after the addition of rhodamine dextran.