Isolation of Mouse Alveolar Type II Cells

Adult mouse lungs were used for the isolation of AT2 cells [11 (link)]. In brief, the lung was perfused with 4 °C PBS. After trachea intubation, dispase II (3 mg/mL, Corning, New York, NY, USA) was injected into the lung, followed by 1% low-melting-point agarose (42–45 °C, Sangon Biotech, Shanghai, China). Then, the lung was incubated in dispase II for 45 min at 37 °C and gently torn in DMEM/F12 + DNase I (0.01%, Sigma, St. Louis, MO, USA). The cell suspension was passed through serial filters (Solarbio, Beijing, China) and incubated with biotinylated antibodies: CD16/32 and CD45 (Miltenyi Biotec, Shanghai, China) for 10 min. After being incubated with 10 µL Streptavidin MicroBeads (4 °C, Miltenyi Biotec, Shanghai, China) for 15 min in dark, the cells were transferred onto plates pre-coated with 1 mg/mL IgG for 2 h, and the unattached cells were collected. The cell purity was detected by flow cytometry with corresponding antibodies.