Isolation and Characterization of Alveolar Macrophages

BALF was collected as described (Li et al, 2013 (link)) and centrifuged at 400g for 10 min to separate the supernatant from cells. The numbers of total cells, neutrophils, and macrophages from BALF were visualized using the Wright–Giemsa staining (Sigma-Aldrich) and counted in a double-blind manner. Isolation of alveolar macrophages from BALF was performed as described in detail earlier (Busch et al, 2019 (link)), and the purity of isolated macrophages was characterized by dual staining with PE-conjugated sialic acid–binding immunoglobulin-like lectin F (Siglec-F) and Alexa Fluor 488–conjugated CD11c (BioLegend) followed by flow cytometry.