Bovine Bronchial Epithelial Cell Culture

Fresh lungs were collected from calves at a local slaughterhouse. Bovine primary bronchial epithelial cells (PBEC) were isolated as previously described [44 (link)] and were expanded in growth medium (BEGM). When the PBEC had reached confluence, 5 × 10⁵ cells were seeded on the apical compartment of Corning Transwell® polycarbonate membranes with 300 µl medium in the apical compartment and incubated for 24 h at 37°C in a humidified 5% CO₂ atmosphere, while the basolateral compartment was filled with 600 µl medium. The transepithelial electrical resistance (TEER) was measured every day by using the Millicell® ERS-2 Voltohmmeter (Millipore) according to the manufacturer’s instructions. Only monolayers with TEER of more than 500 Ω were used for the infection experiments [45 (link)]. Moreover, FITC-labeled 70,000-molecular-weight (Da) dextran (Invitrogen) was added to the apical compartment to measure the integrity of the epithelial barrier. The medium was harvested from the basolateral compartment at different time points, and the fluorescence was determined with a spectrophotometer (Varian Cary Eclipse).