Collagen coated

Cell lines used in this study included Madin-Darby canine kidney (MDCK) cells (American Type Culture Collection, ATCC), LA-4 mouse lung epithelial cells and the RAW264.7 macrophage cell line (ATCC). Resident peritoneal exudate macrophages were obtained from mice as previously described [27 (link)]. Macrophages were seeded into 8-well glass chamber-slides (Lab-Tek), incubated for 2 hours at 37°C and cell monolayers were washed to remove non-adherent cells. The next day, any remaining non-adherent cells were removed and the adherent macrophages used in virus infection assays. Mouse primary lung epithelial cells were prepared as previously described [28 (link)]. Briefly, lungs from mice were digested in 1.5 mg/ml Pronase (Roche, USA), 0.1 mg/ml DNase I (Sigma-Aldrich, USA) for 1 hour at 37°C in 5% CO₂. Single cell suspensions were incubated with purified rat anti-mouse CD45 antibody (BD Biosciences, USA) and epithelial cells negatively enriched with BioMag goat anti-rat immunoglobulin-coupled magnetic beads (Qiagen, USA). Cells were cultured on collagen-coated (MP Biomedicals, USA) plates. To confirm alveolar epithelial cell purity, monolayers were detached with 3 mM EDTA and cells stained with mouse anti-EpCAM, anti-podoplanin (AEC type I) and anti-CD74 (AEC type II) antibodies and examined by flow cytometry.