Measuring Airway Surface Liquid Height in Tracheal Epithelial Cultures

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.