Measuring Airway Surface Liquid in Primary Tracheal Epithelial Cultures

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.