F4 80 bv711

Cells were incubated with UV live/dead stain (Thermo Fisher Scientific, Waltham, MA) and TruStain FcX block (BioLegend, San Diego, CA) prior to Ab staining. IL-10 detection was performed using the IL-10 secretion assay following overnight ex vivo stimulation (Miltenyi Biotec). Cells were stained with Abs to CD4-allophycocyanin Fire 780 (BioLegend), T cell Ig and mucin domain containing 3 (Tim3)-allophycocyanin (BioLegend), CTLA4-allophycocyanin (BioLegend), CD49b-AF647 (BioLegend), CD49b-PE-Cy7 (BD Biosciences), programmed cell death protein 1 (PD1)–allophycocyanin (BioLegend), PD1-BV421 (BioLegend), lymphocyte activating gene 3 (Lag3)–BV785 (BioLegend), F4/80-BV711 (BD Biosciences), and Foxp3-AF488 (BioLegend). Cells were washed in MACS buffer before being analyzed on either a FACSAria SORP (BD Biosciences), LSRFortessa (BD Biosciences), or Attune NxT (Thermo Fisher Scientific) with support of the Cytometry & Imaging Microscopy Core Facility of the Case Comprehensive Cancer Center. Cell sorting was performed on a FACSAria SORP (BD Biosciences). All FACS data were analyzed using FlowJo (Tree Star, Ashland, OR).

Spleen cell suspensions were prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells were then incubated with an antibody at 4 °C for 30 min in the dark in PBS with 2% normal FBS. Flow cytometry was performed using a FACS Fortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), according to standard techniques. To characterize splenic cells, the monoclonal antibodies used spleen cell suspensions prepared after hypotonic lysis of erythrocytes in potassium acetate solution and three washes in complete RPMI medium. For each mouse, splenocytes were quantified using a Malassez counting chamber. Cells’ suspensions were incubated for 20 min with the following antibodies. All antibodies were obtained from Biolegend (San Diego, CA, USA) and used at a dilution of 1:100 unless otherwise mentioned: CD3 FITC, CD4 APC Fire 750 (BD Biosciences, San Jose, CA, USA), CD8 BV 605, CD69 PEDazzle594, CD40 PercPCy5.5, B220 APC, CD44 BV650, MHC II eFluor450 (DAPI) (eBioscience, San Diego, CA, USA), F4/80 BV711 (dilution 1:200), CD11b PercP Cy5.5, CD80 BV421, CD86 FITC, CD206 Alexa Fluor 647 (BD Biosciences, San Jose, CA, USA. Dilution 1:200), Ly6C PECy7.

Gum arabic, activated carbon, 2,7-dichlorofluorescein diacetate (DCFDA), toluidine blue, protease inhibitor cocktail tablets, phosphatase inhibitor cocktail tablets, MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel were purchased from Merck KGaA (Darmstadt, Germany). 25% Aqueous Solution Glutaraldehyde, Paraformaldehyde, Sorensen's Phosphate Buffer, 2% Aqueous Solution Osmium Tetroxide, Ethyl Alcohol, Acetone, Araldite, Dibutyl phthalate (DBP) were purchased Electron Microscopy Sciences (Hatfield, USA). Leukocyte Activation Cocktail with BD GolgiPlug™, FACS antibodies include anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, CD45-APC-Cy7, CD3e-FITC, CD4-V450, CD8-BV510, CD25-BV605, IL-4-PE-Cy7, IFN-γ-PE, FoxP3-AF647, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c--AF700 were purchased from BD (Heidelberg, Germany); Another FACS antibody Anti-mouse-IL-17A-BV650 was purchased from eBioscience (Frankfurt am Main, Germany). Anti-mouse HMGB1, Anti-mouse phospho-p65, anti-mouse phospho-p38, anti-mouse GAPDH and anti-rabbit IgG HRP were obtained from Cell signaling Technology (Frankfurt am Main, Germany). IgE and histamine ELISA kits were purchased from Abcam (Berlin, Germany).