T col

Primary tracheal epithelial cells were isolated and cultured from 6 mice per group as described above. After 14 days in culture, 5000 Nile Red carboxylate-modified fluospheres (2 µm diameter) (ThermoFisher Scientific) dissolved in 2 µl PBS were applied to the center of the apical surface of the cultures that were placed in a microscope incubator (EMBL Heidelberg) at 37 °C and 5% CO₂. MCT rates were determined from the bead velocity after 60 min with a Leica TCS SP8 confocal microscope (Leica Microsystems). Time lapse images were captured in 4 s intervals for 4 min at three different positions of each cell culture insert (T-Col, Costar). Image series were analyzed with the Fiji^{64 (link),65 (link)} plugin Trackmate^{80 (link)} and the simple LAP tracker. Linear, directed transport tracks were identified by a ratio of track displacement to track length > 0.8, minimal displacement of 80 µm and track duration of 16 s. Mean velocity from three different positions was used to calculate a single value for each culture.

For each experiment, freshly excised tracheae were collected and pooled from 10 mice per group. Epithelial cells were isolated and cultured on membranes (T-Col, Costar, Cambridge, MA) under air-liquid interface conditions as described previously [22 (link)], and cultures were studied after reaching confluence (14 days).

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.