Anti f4 80 bm8

For isolation of lung leukocytes, minced lung tissue was digested in RPMI medium supplemented with collagenase (MERCK KGaA) and DNAse (PanReack AppliChem GmbH, Darmstadt, Germany) for 30 min at 37 °C. Single cell suspension was prepared by using 70-μm cell strainers (BD, Heidelberg, Germany) and red blood cell lysis was performed. BAL and lung immune cells were differentiated by flow cytometry. Isolated BAL cells were blocked with anti-CD16/CD32 (BD) and stained with anti-CD11c (N418; ATCC), anti-CD11b (M1/70; eBioscience, Vienna, Austria), anti-F4/80 (BM8; eBioscience), anti-Ly6G (1A8; BD), and anti-Ly6C (AL-21; BD) monoclonal antibodies (mAbs). The FL-1 channel was used to determine autofluorescence of cells. The gating strategy is shown in Additional file 1: Figure S3. Isolated lung cells were blocked with anti-CD16/CD32 (BD), surface stained with anti-CD4 (RM4–5; BD), anti-CD8 (53–6.7; eBioscience), anti CD3 (17A2; eBioscience), and gdTCR (GL-3; BD), fixed and permeabilized with FoxP3 / Transcription Factor Staining Buffer Set (eBioscience) and stained intranuclearly with anti-FoxP3 (FJK-16s; eBioscience) mAbs. All stained cells were acquired using a BD FACS Canto II and analyzed with BD FACSDiva and FlowJo software. For calculation of total cell numbers, CountBright Absolute Counting Beads (Thermo Fisher Scientific) were used.

Five mice per time point per infection were infected with 10⁵ PFU of the described virus. Five uninfected (naïve) mice served as negative controls. Whole lungs were collected from mice, and incubated with Collagenase D (Roche Diagnostics; final concentration: 2 μg/mL) and DNase I (Worthington; final concentration: 40 U/mL) for 30 minutes at 37°C. Single-cell suspensions were obtained from lungs by grinding tissues through a nylon filter (BD Biosciences). Red blood cells (RBCs) in a sample were lysed with RBC lysis buffer (Sigma). Samples were resuspended with PBS containing 2 mM EDTA and 0.5% bovine serum albumin (BSA), and cell number was determined by using a disposable cell counter (OneCell). To block nonspecific binding of antibodies mediated by Fc receptors, cells were incubated with purified anti-mouse CD16/32 (Fc Block, BD Biosciences). Cells were stained with appropriate combinations of fluorescent antibodies to analyze the population of each immune cell subset. The anti-F4/80 (BM8; eBioscience) antibodies were used. All samples were also incubated with 7-aminoactinomycin D (Via-Probe, BD Biosciences) for dead cell exclusion. Data from labeled cells were acquired on a FACSAria II (BD Biosciences) and analyzed with FlowJo software version 9.3.1 (Tree Star).

Antibodies used for flow cytometry analysis of surface marker expression on BMDCs are the following: anti-CD11c (N418); anti-CD40 (1C10); anti-CD80 (16-10A1); anti-CD86 (GL1); and anti-I-A/I-E (M5/114.15.2) (all eBioscience). Antibodies used for flow cytometry analysis of cells from mediastinal LNs are the following: anti-CD19 (eBio1D3); anti-F4/80 (BM8) (eBioscience); anti-CD3ε (145-2C11); anti-CD45 (30-F11); anti-CD11c (N418); and anti-I-A/I-E (M5/114.15.2) (BioLegend). For staining of intracellular protein iNOS (C-11) (Santa Cruz Biotechnology), samples were first fixed with IC Fixation Buffer (eBioscience) and stained in Permeabilization Buffer (eBioscience). Samples were acquired on the BD LSR Fortessa and data analyzed using FlowJo software.

The following mAbs from BD Biosciences were used: anti-CD45 (30-F11), anti-CD8 (53-6.7), anti-CD4 (RM4-5), anti-CD62L (MEL-14), anti-CD90.1 (OX-7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD25 (PC61 or 7D4), anti-ICOS (7E.17G9), anti-GITR (DTA-1), anti-CD103 (M290), anti-Helios (22F6), anti-CTLA-4 (UC10-4F10-11), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD19 (1D3), anti-IA/E (M5/114.15.2), anti-Ly6C (AL-21), anti-Ly6G (1A8). Anti-GFP antibody was purchased from Life Technologies. Anti-CD3 (145-2C11), anti-Foxp3 (FJK-16s), anti-CD44 (IM7), anti-Ki-67 (SOLA15), anti-Nrp1 (3DS304M), anti-NKp46 (29A1.4) and anti-F4/80 (BM8) were purchased from eBioscience, and Foxp3 staining was performed using the eBioscience kit and protocol. Cells were acquired on a BD LSRII and a BD Fortessa X20 cytometers and analyzed using FlowJo software.

To obtain single-cell suspensions, lungs were dissociated with Collagenase D (Roche Diagnostics, Mannheim, Germany; final concentration: 2 μg ml⁻¹) and DNase I (Worthington Biochemical, Lakewood, NJ; final concentration: 40 U ml⁻¹) for 30 min at 37 °C by grinding the tissue through nylon filters (BD Biosciences). Red blood cells (RBCs) were lysed by treatment with RBC lysing buffer (Sigma Aldrich, St Louis, MO). To block nonspecific binding of antibodies, cells were incubated with purified anti-mouse CD16/32 (Fc Block, BD Biosciences, San Diego, CA). Cells were stained with appropriate combinations of fluorescent antibodies to analyse the population of each immune cell subset. The following antibodies were used: anti-CD45 (30-F11: eBioscience, San Diego, CA), anti-CD11b (M1/70: BioLegend), anti-F4/80 (BM8: eBioscience) and anti-CD11c (HL3: BD Biosciences). All samples were also incubated with 7-aminoactinomycin D (Via-Probe, BD Biosciences) for dead cell exclusion. Data from labelled cells were acquired on a FACSAria II (BD Biosciences) and analysed with FlowJo software version 9.3.1 (Tree Star, San Carlos, CA). To isolate Venus-positive and -negative macrophages from lungs, stained cells were sorted using a FACSAria II (BD Biosciences).

Samples were blocked with 5 µg/mL of anti-CD16/CD32 (eBioscience) and incubated with combinations of the following antibodies, which were either directly conjugated to fluors, or biotinylated: anti-Ly6C (HK 1.4, BD Biosciences); anti-Ly-6G (1A8, BD Biosciences); anti-CD11b (M1/70, BD Biosciences); anti-F4/80 (BM8; eBioscience); anti-CD11c (HL3, BD Biosciences); anti-I-A/I-E (M5/114.15.2, Biolegend); anti-CD115 (AFS98, Biolegend); anti- CD45 (30F11, Biolegend); anti-CD64 (X54-5/7.1,BD Bioscience); anti-mMer (MerTK, R&D Systems); anti-Siglec F (E50-2440, eBioscience); Ki67 (B56, BD Biosciences, used in conjunction with elements of the BrdU detection kit for cell permeabilization). Strep-PECy7 (BD Biosciences) was used to detect biotinylated antibodies. Cells were stained with LIVE/DEAD (Invitrogen) before analysis. Data were acquired on a Canto II (BD Biosciences) and analyzed with FlowJo v.8.8.6 (Tree Star, Inc.). Cells were sorted on a BD FACSAria (BD Biosciences). For morphologic characterizations, sorted cells were prepared on slides by cytocentrifugation at 1000 RPM for 5 min, and stained with HEMA-3 (Fischer Scientific).