Lung Immune Cell Profiling Post-Influenza

BALF was collected on days 0, 7, and 8 post-influenza virus infection by flushing the lungs twice with 1 ml PBS. BALF was centrifuged, and the cell pellet was used for cell analysis. Cells were treated with Fc block (BD Biosciences) and surface stained with the following antibodies (at 1 µg/ml): anti-CD11b (clone M1/70, BioLegend), anti-CD11c (clone HL3; BD Biosciences), anti-F4/80 (clone BM8; BioLegend), and anti-Ly6G (clone 1A8; BioLegend). Data were acquired for equivalent numbers of cells on a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 9. Cell types were defined as previously described (24 (link)). Briefly, neutrophils were defined as CD11b^hi Ly6G^hi cells, and alveolar macrophages were defined as CD11c^hi F4/80^hi CD11b⁻ cells.