Anti mhcii pe

On the third day of infection, peritoneal cells from WT mice infected with 1 × 10⁷ Nc-1 tachyzoites were collected and stained for phenotypic characterization and Dectin-1 expression. Briefly, mice were euthanized, and their peritoneal cavities were washed with ice-cold PBS. The suspension was then centrifuged at 400 × g, at 4°C, for 10 min. The cell pellet was resuspended in PBS with 5% of normal rabbit serum, at room temperature, for 15 min, prior to incubation with the appropriate antibodies: anti-CD11b-APC-Cy7, anti-CD11c-FITC, anti-MHCII-PE, anti-CD11c-V450, anti-CD19-APC-Cy7, anti-CD3-Pacific Blue, anti-CD49b-APC (BD Biosciences), and anti-Dectin-1-PE (R&D Systems, Minneapolis, MN, USA). Cells were incubated with primary antibodies conjugated to the different fluorochromes for another 30 min, at room temperature. After washing, cells were suspended in PBS with 3% formaldehyde, read by flow cytometry (FACSCantoII, BD Biosciences), and analyzed using dedicated software (FlowJo X, Tree Star Inc., Ashland, OR, USA).

DEAE-52 cellulose was obtained from GE Healthcare (Uppsala, Sweden). The monosaccharides, comprising ribose (Rib), glucuronic acid (GlcA), glucose (Glc), galactose (Gla), mannose (Man), arabinose (Ara), rhamnose (Rha) and xylose (Xyl), lipopolysaccharide (LPS), active carbon, trifluoroacetic acids (TFA), and FITC-Dextran, were purchased from Sigma-Aldrich (St Louis, MO, USA). T-series dextrans (T-10, T-40, T-70, T-500 and Blue dextran) were purchased from Solarbio (Beijing, China). Fetal bovine serum (FBS) and Penicillin-streptomycin were purchased from MRC (Changzhou, China). Medium RPMI-1640 and phosphate-buffered solution (PBS) were purchased from Gibco (Grand Island, NY, USA). Granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from PeproTech (Rocky Hill, NJ, USA). The other chemical reagents were purchased from Tianjin Fuchen (Tianjin, China).
The antibodies for flow cytometry comprising anti-CD40-APC, anti-CD86-APC, anti-CD11c-FITC, and ELISA kits including tumor necrosis factor-α (TNF-α), IL-1β, and IL-12p40 were purchased from Elabscience (Wuhan, China). Anti-MHC-I-FITC, anti-MHC-II-PE, and anti-CD11c-PE were bought from BD Biosciences (San Diego, CA, USA).

Mouse lung tissue was digested with collagenase to generate a single-cell suspension. Lung cells or cultured cells were stained with CD11c (anti-CD11c-PE-Cy5.5, e Bioscience, USA), MHCII (anti-MHCII-PE, BD Pharmingen, USA) and GITRL (anti-GITRL-BV421, BD Pharmingen, USA) to identify CD11c⁺MHCII⁺GITRL⁺ DCs. For the analysis of Tregs, lung cells or cultured cells were first stained for CD4 (anti-CD4-APC-Cy7, BD Pharmingen, USA) and CD25 (anti-CD25-PE, BD Pharmingen, USA) and then fixed and permeabilized for intracellular Foxp3 (anti-Foxp3-APC, e Bioscience, USA) staining. For detecting Th1, Th2 and Th17 cells, lung cells or cultured cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μg/ml) for 6 h in the presence of Golgi Stop solution. After stimulation, cells were incubated with surface markers for CD4 (anti-CD4-FITC, BD Pharmingen, USA), permeabilized and stained for the intracellular cytokines IL-4 (anti-IL-4-APC, BD Pharmingen, USA), IFN-γ (anti-IFN-γ-BV421, BD Pharmingen, USA) and IL-17 (anti-IL-17-PE, BD Pharmingen, USA). Data were acquired using FACS Canto II (BD Biosciences, USA) and analyzed with FlowJo software.