Following sacrifice, mouse alveolar epithelial cells were separated from the lungs of mice in each group. First, 20 ml PBS was used to flush blood from the lung vasculature. Subsequently, a 1-2-mm section of lung tissue was excised and cut into 1-mm3 pieces on ice. To disperse and culture fresh tissue, tissues were cultured in DMEM (3 ml; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 20% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) in 25 ml uncoated culture flasks with 5% CO2 at 37°C for 48-52 h. Tissue explants were removed and cells were maintained in DMEM (3 ml) supplemented with 20% fetal calf serum. After monolayers formed, cells were purified (1×105/ml) and cultured in DMEM supplemented with 10% fetal calf serum. Following culture for 3 days, cells were used for subsequent experiments. Alveolar epithelial cell morphology and proliferation were observed using an inverted light microscope (magnification, ×200) to determine cell viability and purity (data not shown). Alveolar epithelial cell structure was observed by transmission electron microscopy using a JEM-2000EX transmission electron microscope (magnification, ×17,000) according to the aforementioned protocol.

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