Isolation and Characterization of Alveolar Macrophages

Mice AMs were obtained as previously described [68 (link)], and their phenotype was analyzed. Briefly, bronchoalveolar lavage fluid (BALF) from C57BL/6 mice was collected by inserting a 22G needle cannula into trachea. Ice-cold PBS was slowly instilled into the lung, and then the lavage fluid was collected as BALF. The BALF was centrifuged and cell pellets were collected. Cells were cultured in RPMI 1640 complete medium for 6 h. Then, adherent cells were collected as AMs. For identification of AMs, cells were stained with antibodies of CD11c (Miltenyi, cat no: 130122939, Bergisch Gladbach, Germany), CD11b (Miltenyi, cat no: 130113806, Bergisch Gladbach, Germany), CD45 (BD, cat no: 561018, Franklin Lakes, NJ, USA) and siglec-F (Miltenyi, cat no: 130123816, Bergisch Gladbach, Germany) and analyzed by flow cytometry. For detection of CD206 and CD80 expression in AMs, these cells were stained with antibodies of CD206 (Biolegend, cat no: 141712, San Diego, CA, USA) and CD80 (Miltenyi, cat no: 130102883, Bergisch Gladbach, Germany), and analyzed through flow cytometry.