We developed a gating strategy (Fig. 1 and Table 1) and fluorophore panel (Supplemental Table S2) for spectral flow cytometry that is compatible with heterogeneous AF and characterizes B cells, T cells (cytotoxic T, T helper, and γδ T cells), NK cells, macrophages (alveolar and recruited), monocyte-macrophages, monocytes (Ly6Clo and Ly6Chi), dendritic cells (CD103+ and CD11b+), neutrophils, and eosinophils in the pulmonary tissues during influenza infection. The gene discussed is ITGAM; the disease is influenza.