Fc blocker cd16 cd32

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).

Spleens from uninfected and infected C57BL/6 and Prf1^-/- mice were embedded in OCT (Sakura Finetek, Torrance, CA), frozen in dry ice, stored frozen at -80°C and incubated at -20°C for 30 minutes before cryostat sectioning. Frozen sections (10) μm were cut and adhered to Poly-Prep slides (Sigma-Aldrich, St. Louis, MO), air dried and fixed in ice cold acetone for 10 minutes. Slides were blocked with 5% bovine serum albumin (BSA) and Fc blocker CD16/CD32 (eBioscience) for 30 minutes and stained with biotin-conjugated antibody MOMA-1 at room temperature for one hour. Sections were washed and treated for one hour at room temperature with Avidin FITC and antibody B220. Stained sections were washed, air dried at room temperature and mounted with ProLong Gold antifade reagent (Life Technologies) and covered with glass coverslips (Corning). All the slides were read on a Zeiss MOT200 inverted microscope with a Zeiss apotome at 20x magnification.

After PBMCs were thawed, the Fixable Viability Dye Kit (eBioscience, San Diego, CA) was used to assess cell viability by FACS analysis (>90% in all samples). 500,000 viable singlet-events were sorted per sample using a Sony SH800 Cell Sorter (Sony Biotechnology, San Jose, CA). The sorted cells were incubated with Fc blocker (CD16/CD32, eBioscience) for 10 min, after which each sample was incubated with TotalSeq Hashtag antibody tags to enable multiplexing and subsequent deconvoluting. After Hashtag staining, the cells were pooled into four pools of five samples, each sample contributing equally to their respective pool. The cells were counted manually by light microscopy and Neubauer chambers. Each pooled sample was then incubated for 30 min with our TotalSeq oligo-conjugated antibody panel for later surface protein marker quantification. An overview of all antibodies that were used in the study is shown in Supplementary file 2.