Ly6g fitc

Peripheral blood mononuclear cells, splenocytes, or peritoneal macrophages at a concentration of 1 × 10⁶ cells were centrifuged at 192× g for 7 min at 4 °C. Subsequently, samples were incubated with anti-CD16/CD32 (BD Biosciences, San Jose, CA, USA) antibodies for 10 min to block the Fc receptors. After this, PBMCs and splenocytes were incubated with the rat anti-mouse antibodies CD11b-APC, Ly-6C-PE, and Ly-6G-FITC (BD Biosciences, San Jose, CA, USA), while peritoneal macrophages were stained with rat anti-mouse antibodies CD45-PerCP-Cy5.5, CD11b-APC, CD36-PE, CD86-FITC, and Ly6G/Ly6C-APC-Cy7 (BD Biosciences, San Jose, CA, USA) for 30 min at 4 °C in the dark. Subsequently, cells were centrifuged and resuspended in PBS supplemented with 2% FBS. Samples were analyzed in BD LSR Fortessa cytometer with FACSDiva v8.0.1 software (BD Biosciences, San Jose, CA, USA).

A nonparenchymal cell fraction from whole liver extracts was isolated upon collagenase and mechanical digestion followed by Percoll (GE Healthcare Life Sciences) gradient centrifugation as previously described [5 (link)]. In parallel, blood samples were collected in EDTA-containing tubes and treated with red blood cell lysis buffer (PharmLyse, BD Biosciences, Germany). Upon removal of red bodies and centrifugation, immune cells were incubated with fluorochrome-conjugated antibodies and characterized according to two different panels, a myeloid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD11b-BV711 (101242, BioLegend), F4/80-APC (17-4801-82, eBiosciences), MHC2-Alexa700 (107622, BioLegend), CD11c-PE-Cy7 (25-0114-81, eBiosciences), and Ly6G-FITC (551460, BD Pharmingen) and a lymphoid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD3-PE-Cy7 (25-0031-82, eBiosciences), CD4-FITC (11-0041-85, eBiosciences), CD8-PerCpCy5.5 (126610, BioLegend), and NK1.1-BV711 (108745, BioLegend). Labeled cells were then subjected to flow cytometry using a BD Canto II (BD Biosciences) and relative cell numbers were analyzed using FlowJo software (Tree Star).

PleCs and BAL cells were analyzed. Firstly, red blood cells were lysed by hypotonic shock. The cell suspensions were then centrifuged at 250 g for 8 min at 4°C, diluted in 1 ml PBS with 2% FCS and counted in PBS with 0.04% trypan blue by using a haemocytometer (KOVA Glasstic Slide). Cells were incubated 20 min with CD16/CD32. Proportions of the different leukocyte populations were determined by flow cytometry using the following rat anti-mouse antibodies: anti- F4/80-APC (dilution 1:200; eBioscience, clone BM8), anti-SiglecF-PE (dilution 1:200, BD Bioscience, clone E50-2440) and Ly6G-FITC (dilution 1:200, BD Bioscience, clone 1A8). Flow cytometry acquisition was performed using a FACSVerse flow cytometer running the FACSuite software (BD Biosciences). Doublets and debris were excluded. Analyses were performed with FACSuite software.

Each mouse lymph nodes and spleen were minced separately and passed through a 70 μm cell strainer to obtain single-cell suspensions. Single cells from the draining lymph nodes were used for the detection of innate immune cells and were stained with the following monoclonal antibodies: F4/80-PerCP-Cy 5.5, CD11c-PE, CD11b-APC, and Ly6G-FITC (BD Biosciences, USA). The γδT and Th17 cells from the spleen samples were detected with the following fluorescent antibodies: CD45-PerCP-Cy 5.5, γδT-PE, CD4-APC, and IL-17A-PE (BD Biosciences, USA). For the surface antigen staining, cells were incubated with the antibodies for 30 min at room temperature (RT) after washing the cells with PBS. To stain the intracellular cytokines, the cells were stimulated with a cell stimulation cocktail (eBiosicence) for 4 h. After that, the cells were stained for surface antigens and then fixed with BD Cytofix buffer, permeabilized by using Perm/Wash reagent (BD Biosciences) and then stained with anti-IL-17A antibodies. The cells were acquired using an LSR II Flow Cytometer (BD Biosciences), and the data were analyzed using the Flow Jo software version 7.0 (Tree Star, California, USA).

Mouse blood was collected 1 week after sponge implantation from vena saphena in Ethylenediaminetetraacetic acid (EDTA) tubes. Red blood cells were lysed after which cells were stained with a myeloid panel of antibodies comprising Ly6G-FITC (561105, BD Biosciences), Ly6C-BV605 (563011, BD Biosciences), and CD11b-BV711 (563168, BD Biosciences) for 30 minutes at 4°C. Cells were washed and resuspended in DAPI (Invitrogen) before acquisition on a five laser BD LSR Fortessa and analyzed with DIVA (BD Biosciences).
To determine intracellular ROS levels, cells were stained with Ly6C-BV605 (563011, BD Biosciences) and CD11b-BV711 (563168, BD Biosciences) for 30 minutes at 4°C. After washing, cells were stained with H2-DCFDA and analyzed by flow cytometry as described above.

Immunophenotypic analyses of splenoctyes from animals were assessed by flow cytometry. Antibodies to stain for MDSC were CD11b-APC (Clone M1/70; BD Biosciences), Ly6G-FITC (Clone 1A8; BD Biosciences), Ly6C-PE (Clone AL-21; BD Biosciences); for dendritic cells were CD11c (Clone HL3; BD Biosciences); for B cells B220-APC (Clone RA3–6B2; BD Biosciences), CD3-FITC (Clone 145–1011; BD Biosciences). T regulatory cells with a phenotype of CD4⁺CD25⁺FoxP3⁺ were evaluated using a commercially available kit (eBiosciences, San Diego, CA). For T cell activation markers, cells were stained with antibodies specific for CD4-PE-Cy7 (Clone RM4–5; BD Biosciences), CD8-PE-Cy7 (Clone 53–6.7; BD Biosciences), CD62L-PE (Clone MEL-14; BD Biosciences), and CD44-Bv650 (Clone IM7; Biolegend). To determine Th1 and Th2 phenotypes, cells were stained using fluorochrome conjugated antibodies targeted CXCR3-PE-Cy7 (Clone CXCR13–173; Biolegend), CCR4-PE (Clone 2G12; Biolegend), and CCR6-APC (Clone CK4-L3; BD Biosciences). Cells were incubated on ice for 30 minutes, washed, and fixed in PBS containing 1% formalin for flow cytometric analysis on a LSRII flow cytometer (BD Biosciences).