Cd49b pe cy7

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).

Flow cytometry was performed as previously described [38 (link)]. Briefly, cells were incubated with LIVE/DEAD Fixable cell stain (Invitrogen), then stained for 20 min with: MHCII-PacificBlue, F4/80-APC (Biolegend), CD4-AF700, CD8-PacificBlue, CD25-APC, CD86-APC, CD49b-Pe-Cy7 (eBioscience), CD11b-PE-Cy7, CD45R/B220-PerCP-Cy5.5 (BD-Pharmingen). For intracellular staining, the samples were stimulated for 3 h (100 ng/ml phorbol 12-myristate 13-acetate, 1 μg/ml ionomycin; monensin added after 1 h). Cells were fixed and permeabilized with Cytofix/Cytoperm (BD), then stained with IL-10-FITC, IFN-γ-PerCP-Cy5.5, IL-17-PE, FoxP3-AF488 (BD-Pharmingen) antibodies before analysis on LSRII flow cytometer (BD), collecting 100,000 live events. Data was analyzed using FlowJo (v7.6.5, Tree Star, Ashland, OR, USA).