5, or 0.0625 HAU) was given. Control mice were given normal egg allantoic fluid i.n. for mock infection. Mice were monitored for weight daily and euthanized when moribund. Lungs were removed aseptically, and perfused through the right ventricle with 5 mL HBSS to remove peripheral blood cells. To obtain mononuclear cells from lung tissue, the lungs were minced into 2–3 mm sections with scissors and resuspended

in DMEM medium supplemented with 10% FBS, 1–2 mg/mL collagenase (Sigma-Aldrich), 50 U/mL DNase (Sigma-Aldrich), HEPES, and antibiotic antimycotic solution (Sigma-Aldrich). The tissues were incubated at 37°C for 60 min with gentle vortexing at 200 rpm. Lung portions Hedgehog inhibitor were then crushed through 40 μm basket filters and the remaining erythrocytes lysed with lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.4) and washed with PBS. The resulting cell suspensions were used for flow cytometric experiments or further cell purification. In some experiments, lymphocytes were purified from lung preparations by Percoll continuous gradient,

as previously described [49], prior to cytometric analysis of NK cells. The following purified mouse antigen specific conjugated or unconjugated antibodies: INK 128 order CD16/CD32, CD3-FITC, KLRG1-FITC, NKG2A-FITC, IFNγ-FITC, CD244(2B4)-FITC, Rat IgG2a k Isotype control FITC, NK1.1-allophycocyanin, Mouse IgG2a k Isotype control allophycocyanin, IFN-γ-allophycocyanin, KLRG1-allophycocyanin, CD3e allophycocyanin-eFluor780, CD11b-PE, NK1.1-PE, Ly49C/I-PE, CD107a-PE, NKp46-PE, Rat IgG2a k Isotype control PE, CD107a-PerCP-eFluor710, Rat IgG2a k Isotype

control PerCP-eFluor710, CD3-PerCP-eFluor710, NKp46-PerCP-eFluor710, CD27-PerCP-eFluor710, IFN-γ-PerCP-Cy5.5, Rat IgG1 k Isotype control PerCP-Cy5.5, CD122-eFluor450, and Rat IgG2b k Isotype control eFluor450 were purchased from eBioscience (San Diego, CA, USA). CD127-PE-Cy7 was purchased from BD Biosciences. The above-mentioned antibodies were used for FACS analysis in this study. Cells were suspended in buffer comprised of PBS containing 1% FCS plus 0.09% NaN3, followed by incubation with anti-CD16/CD32 mAb and then stained with mAbs specific for cell surface markers for 30 min at 4°C. For intracellular however staining, cells were fixed with 4% paraformaldehyde fixative and then stained for 30 min in 0.1% saponin, 0.05% NaN3 in HBSS at room temperature. Events were collected on a BD FACSCanto II, and the data was analyzed using BD FACSDiva software. In order to deplete NK cells in mice with influenza infection, mice (4 months old) were i.v. injected with 50 μL anti-asialo GM1 [34] (Wako Chemicals) into the tail vein once every 5 days, starting on day 0. As previously described [50, 51], anti-NK1.1 antibodies were purified from the supernatant of PK136 hybridoma cell culture (American Type Culture Collection, Manassas, VA), and i.v. injected into mice (500 μg/injection) on the same schedule. Control mice were treated with PBS.