Gr1 fitc rb6 8c5

BAL and lung cells were treated with RBC lysing buffer (Sigma). Lungs were dissociated using a Lung Dissociation Kit (Miltenyi Biotec) and Gentle MACS (Miltenyi Biotec). LIVE/DEAD cells were stained with Fixable Aqua Dead Cell Staining Kit (Thermo Fisher Scientific), and after Fc block with the 2.4G2 mAb (eBioscience), cells were stained with the following Abs: DR3-PE (4C12; BioLegend), SiglecF (E50-2440; BD Biosciences), CD11b (M1/70; BD Biosciences), CD11c (HL3; BD Biosciences), Ly6G (1A8; BioLegend), ST2-PE (101001PE, MD), CD90.2 (30-H12; BioLegend). For lineage markers for ILC2 staining, the following Abs were used: CD3-FITC (145-2C11; eBioscience), CD4-FITC (GK1.5; eBioscience), CD8- FITC (5H-10-1; BioLegend), CD19-FITC (1D3; BioLegend), NK1.1-FITC (PK136; BioLegend), CD11b-FITC (M1/70; BioLegend), CD11c-FITC (HL3; BD Biosciences), and Gr1-FITC (RB6-8C5; BioLegend). Flow cytometry analysis was performed on a Fortessa (BD Biosciences), and data were analyzed using FlowJo Software (version 10; FlowJo, Ashland, OR). Live CD45+ lung immune cells were separated into T cells (CD3+, CD90.2+), macrophages (CD11b+, CD11c+ SiglecF+), DCs (CD11c+ MHC class II+), neutrophils (GR1+, CD11b+ SigF-), eosinophils (Ly6C+, SigF+, CD11c-) and ILC2 (Lin^- Thy1.2⁺ ST2⁺Sca1⁺).

Bone marrow and spleen cells were stained for flow cytometry as previously described (Mayer et al., 2017 (link)). Gating strategy for BM erythroid cells was described in the Supplementary Figure 1. Cells were stained with the following antibodies: CD45-FITC (30-F11, eBioscience), CD11b-FITC (M1/70, eBioscience), Gr-1-FITC (RB6-8C5, eBioscience), Ter119-APC (TER119, Biolegend) and CD44-PE (IM7, Biolegend), F4/80-APC (BM8, Biolegend), Gr-1-FITC (RB6-8C5, Biolegend), CD3-PE (145-2C11, Biolegend), Ly6C-PE (HK1.4, Biolegend), CD106-PE (429, Biolegend), CD45R/B220-APC (RA3.682, Biolegend). Cells were analyzed using AccuriC6 flow cytometer and data were analyzed using FlowJo software (v10.7.1).