Ly6g alexa700

Cells in BAL fluid were stained with CD3 FITC, CD11c PercP, Siglec F Alexa 647, CD11b PE-Cy7, viability dye APC Cy7 (all BD Biosciences, San Jose, CA, USA), Ly6G Alexa700 (Biolegend, San Diego, CA, USA), MHCII PE, and CD45 PE-eFluor610 (eBiosciences, San Diego, CA, USA) in the presence of Fc blocker (CD16/CD32, eBiosciences). Single cell suspensions from lungs were stained with CD4 FITC, CD45 PerCP-Cy5.5 (eBiosciences), GATA-3 Alexa 647, and viability dye APC-Cy7 (BD Biosciences). The following markers were used for the analysis of ILCs in lung tissue: Lineage (Lin) markers including CD3e, CD19, GR1, B220, Ter119, FcaR1 (all FITC, Biolegend), CD45 Alexa700, CD90 PE, ST2 Brilliant Violet 421 (Biolegend), CD49b PE-Cy7 (eBiosciences) and CD3 Percp-Cy5.5 (BD biosciences). Mediastinal lymph nodes (mLN) cells were stained with CD45 PerCP-Cy5.5, CD4 FITC, GATA-3 Alexa 647 (BD Biosciences) and IL-4 APC (Biolegend). For intracellular/intranuclear staining, cells were permeabilized and fixed using a FOXp3 Staining Buffer set (eBioscience) and subsequently stained with the appropriated markers. All appropriate Fluorescence Minus One (FMO) controls were used. Data were collected on a BD Biosciences Canto II flow cytometer or BD FACSAria™ III and analyzed using FlowJo software (Treestar, Palo Alto, CA, USA).

Mouse peritoneal cell suspensions were stained with the following fluorescence-conjugated mAbs: CD3-BUV737 (17A2) (BD Biosciences, Franklin Lakes, NJ, USA), CD4-FITC (GK1.5) (BD Biosciences), CD8-V500 (53–6.7) (BD Biosciences), CD11b-e450 (M1/70) (Invitrogen), CD11c-PerCP-Cy5.5 (HL3) (BD Biosciences), CD19-APC (1D3) (Invitrogen), CD49b-PE (DX5) (BD Biosciences), F4/80-PE-Cy7 (BM8) (Invitrogen), and Ly6G-Alexa700 (BioLegend, San Diego, CA, USA). Non-viable cells were excluded using fixable viability dye eFluor780 or 506 reagent (Thermo Fisher Scientific). Flow data were collected using a FACS Fortessa X-20 flow cytometer (BD Biosciences), and results were analyzed using FlowJo version 9 software (TreeStar, Ashland, OR, USA).