Eosinophil Identification by Flow Cytometry

Flow cytometry and FACS were performed as described previously [27 (link),29 ]. Briefly, cells isolated from lung tissue as described above were pelleted and resuspended at 10⁷ cells/mL in PBS with 0.1% BSA. Non-specific antibody binding was blocked with anti-CD16/CD32 followed by a master mix antibody cocktail (50 μL per 10⁷ cells) that included 10 μL of each of the following antibodies: anti-CD45-eF450 (clone 30-F11; eBioscience), anti-CD11c-AF700 (clone N418; eBioscience), anti-Gr1 (Ly-6G and Ly-6C)-APC (clone RB6-8C5; BD Biosciences), anti-Siglec F-PE (clone E50-2440; BD Biosciences) and anti-MHCII (I-A/I-E)-PE-CY-7 (clone M5/114.15.2; eBioscience). Cells were incubated with antibodies at 4°C for 30 min while protected from light, then pelleted and resuspended in PBS with 0.5% BSA at 10⁸ cells/mL. Samples were evaluated and sorted using a FACSAria Fusion cytometer (BD Biosciences). Eosinophils were identified as CD45⁺/CD11c^-/GR1^lo/MHCII^-/Siglec F⁺ cells. Additional details are provided in S1 Appendix.