Anti cd11c clone hl3

Naïve and infected footpads were harvested every 2 weeks after infection with L. major or L. major RFP. Samples were digested with 62.5µg/mL liberase TL (Roche, Mannheim, GE) and 0.5mg/mL of DNAse I (Sigma) in RPMI 1640 (GIBCO) for 1h30min at 37°C. After digestion, the enzymatic reaction was terminated by adding RPMI 1640 containing 5% of FBS (Cultilab). The samples were homogenized and centrifuged for 10 minutes at 400xg at 4°C. The pellet was washed, cells were counted, and 10⁶ cells were plated in 96-well plate (Corning, New York, NY, USA). Surface staining was performed for 20min at 4°C using Fc block Fc-γ III/II CD16/32 (clone 2.4G2; BD), anti-Ly6G (clone 1A8; BD); anti-Ly6C (clone HK1.4; BD); anti-F4/80 (clone BM8; eBioscience, San Diego, CA, USA); anti-CD11c (clone HL3; BD); anti-CD11b (clone M1/70; BD); anti-CD8 (clone 53-6.72; BD); anti-CD3 (clone 145-2C11; BD); anti-CD19 (1D3; BD); anti-CD4 (clone GK1.5; BD). For intracellular staining samples were fixed and permeabilized using Fix/Perm Kit from BD, then stained with anti-NOS2 (clone CXNFT; eBioscience). Samples were acquired using FACs Canto II (BD).

BALF was collected on days 0, 7, and 8 post-influenza virus infection by flushing the lungs twice with 1 ml PBS. BALF was centrifuged, and the cell pellet was used for cell analysis. Cells were treated with Fc block (BD Biosciences) and surface stained with the following antibodies (at 1 µg/ml): anti-CD11b (clone M1/70, BioLegend), anti-CD11c (clone HL3; BD Biosciences), anti-F4/80 (clone BM8; BioLegend), and anti-Ly6G (clone 1A8; BioLegend). Data were acquired for equivalent numbers of cells on a FACSCanto II flow cytometer (BD Biosciences) and analyzed with FlowJo software version 9. Cell types were defined as previously described (24 (link)). Briefly, neutrophils were defined as CD11b^hi Ly6G^hi cells, and alveolar macrophages were defined as CD11c^hi F4/80^hi CD11b⁻ cells.

For the in vivo interaction experiment, C57BL/6 mice (CD45.1) were irradiated at 900 Rads. Each irradiated animal received 10×10⁶ bone marrow cells by i.v route, isolated from GREAT IFN-γ GFP reporter mice and REX3 CXCL10-BFP and CXCL9-RFP reporter mice (CD45.2). Twelve weeks after the transference, mice were infected with T. cruzi and, on day 12 after infection, spleens were harvested, then fixed with 1% of PFA, and labeled with DRAQ5 (BD, Pharmingen) and anti-CD8 (clone 53–67, BD). For experiments with REX3 mice, spleen cells were harvested on day 12 after infection, then fixed with 1% of PFA and labeled with: anti-CD8 (clone 53–67, BD), anti-CD11b (clone M1/70, ebioscience), anti-CD209a (clone MMD3, ebioscience), anti-CD317 (pDCA-1, Miltenyi Biotec), anti-CD11c (clone HL3, BD), and DRAQ5 (BD, Pharmingen). The samples were acquired from ImageStream Ammis, and we used the Ideas software to perform the analysis. The double cells were selected based on aspect ratio versus cell area [23 (link)].

Single-cell suspensions were prepared from the blood and brain tissues of control or surgery group mice at postoperative day 2. Brain tissues were dissociated by MACS Neural Tissue Dissociation Kit (Miltenyi Biotec GmbH, Teterow, Germany) according to the protocol of the manufacturer. The cells were incubated with Fixable Viability Stain 510 (BD PharMingen, San Diego, CA, United States) for 15 min at room temperature protected from light. After that, these cells were stained with fluorochrome-conjugated antibodies. The following mouse antibodies were used in this study: anti-CD45 (clone 104, #560694, BD Biosciences, San Diego, United States), anti-CD11b (clone M1/70, #561098, BD Biosciences, San Diego, United States), anti-Ly6G (clone 1A8, #127614, Biolegend, United States), anti-Ly6C (clone HK1.4, #128007, Biolegend, United States), anti-F4/80 (clone T45-2342, #565411, BD Biosciences, San Diego, United States), anti-CD3 (clone 145-2C11, #551163, BD Biosciences, San Diego, United States), anti-CD19 (clone 1D3, #561738, BD Biosciences, San Diego, United States), anti-NK1.1 (clone PK136, #45-5941-80, Invitrogen, Carlsbad, United States), and anti-CD11c (clone HL3, #561022, BD Biosciences, San Diego, United States). All acquisitions were performed using BD Biosciences FACSVerse™ and analyzed using FlowJo 10.4 software.

Cells were stained in 1X PBS containing 2% FBS and 2 mM EDTA for 30 min at 4°C and then fixed with 2% PFA for 15 min at 4°C. Single-cell preparations were stained with antibodies to the following markers: anti-Ly6C (clone HK1.4, BioLegend), anti-IgA (clone mA-6E1, eBioscience), anti-Ly6G (clone 1A8, BD), anti-SiglecF (clone E50-2440, BD), anti-CD11c (clone HL3, BD), anti-CD45 (clone 30-F11, BioLegend), anti-IA/IE (clone M5/114.15.2, BD), anti-B220 (clone RA3-6B2, BioLegend), anti-CD138 (clone 281-2, BioLegend), anti-CD64 (clone X54-5/7.1, BD), anti-CD11b (clone M1-70, BD) and anti-CD5 (clone 53-7.3, BD). Dead cells were excluded from analysis using the viability dye BD Horizon Fixable Viability Stain 510 (BD). Data were acquired in an LSRFortessa X20 cytometer (BD Biosciences) and analyzed using FlowJo v10.0.7 software (BD Biosciences).