Siglec f bv711

BAL leukocytes, tissue-isolated leukocytes or in vitro-stimulated cDCs were stained with TruStain FcX anti-mouse CD16/32 antibody (BioLegend, San Diego, CA, USA) and CD45-APC-Cy7, CD103-PE, CD11b-PeCy7, CD11c-BV711, I-A/I-E (MHC II)-Pacific Blue, I-A/I-E (MHC II)-PERCP, CD172a (Sirpα)-APC-Cy7, CD19-biotin, CD90.2-biotin, IRF4-PE, Ly-6G-PE, XCR1-APC, CD8α-APC-Cy7, Lineage antibody cocktail-Pacific Blue, CD135 (FLT3)-biotin, Streptavidin-PERCP (BioLegend), NK1.1-biotin (ablab, Vancouver, BC, CA), CD11c-APC, Siglec-F-BV711 (BD Biosciences, San Jose, USA), IRF8-APC (Miltenyi Biotec, Bergisch Gladbach, Allemagne) and Streptavidin-Pe-Cy7 (eBioscience, Thermo Fisher Scientific), GM–CSFRα-APC (R&D system, Minneapolis, MN, USA). Total, neutrophils and macrophages BAL number were determined using precision count beads (BioLegend). Intracellular staining was performed using the True-Nuclear™ Transcription Factor Buffer Set (BioLegend) according to the manufacturer’s instructions. Cells were analyzed using a BD LSR Fortessa cytometer (BD Biosciences) and FlowJo software V10 (BD, Franklin Lakes, NJ, USA). Mean fluorescence intensity (MFI) data were analyzed as Δ MFI, which corresponds to the MFI of the antigen-positive population minus the MFI of the fluorescence minus one (FMO) control of this population.

Bone marrow-derived mEOSs were stained with anti-mouse CD45-APC-Cy7 (BioLegend); CD11b-Pe-Cy7 (BD Biosciences, San Jose, CA, USA); CCR3-Pe-Cy7 (BioLegend); CD44-Pe-Cy7 (BioLegend); CD62-L-Pe-Cy7 (BD Biosciences); Siglec-F-APC-Cy7 (BD Biosciences); CD11c-Pe-Cy7 (BioLegend); and MHCII-Pe-Cy7 (BioLegend). Airway mEOSs were stained with CCR3-Pe-Cy7, CD45-APC-Cy7 and NK1.1-biotin (Ablab, Vancouver, Canada); CD90.2-biotin (BioLegend); CD19-biotin (BioLegend); Siglec-F-BV711 (BD Biosciences); CD11c-Pacific Blue (BioLegend); and Ly-6G-PE (BioLegend). Cells were analyzed by using a BD LSRFortessa cytometer (BD Biosciences) and FlowJo software V10 (BD, Franklin Lakes, NJ, USA).