Flow Cytometry Analysis of BALF Cells

BALF cell composition was determined by flow cytometric analysis of recovered lavage cells in suspension and stained with surface markers. In brief, BALF was centrifuged for 2 min at 2000 rpm, the supernatant removed, and the cell pellet resuspended and washed in 1 ml of FACS buffer (Phosphate Buffered Saline, 5% fetal bovine serum, 0.05% sodium azide). The washed pellet was resuspended and stained in a solution containing FACS buffer with 10% rat serum, Fc-receptor blocking antibody (clone 24G2) and the following antibodies [BD BioScience]: rat-anti-mouse Ly-6G-PECy7, rat-anti-mouse Siglec-F-PE, pan-leukocyte rat-anti mouse CD45-PerCP, and rat-anti-mouse CD11c-APC. Neutrophils were defined as CD45hi Ly6G+, eosinophils were defined as CD45hiLy6G− CD11c− SiglecF+, and alveolar macrophages were defined as CD45hi Ly6G− CD11c+SiglecF+. Flow cytometry was performed on a Guava EasyCyte 8HT (EMD Millipore).