Apc conjugated anti cd11c

Zymosan A (ZyA; Alfa Aesar), DMSO (Tocris Bioscience), Rapamycin (Adipogen), 6-Diazo-5-oxo-L-norleucine (DON; Sigma-Aldrich), Torin1 (Tocris Bioscience), compound 968 (C968; Calbiochem) were purchased. In vitro experiments used the carboxyfluorescein diacetate succinimidyl ester (CFSE) cell proliferation kit (Invitrogen), RPMI 1640 (Wako Pure Chemical Industries), fetal bovine serum (FBS; MP Biomedicals), 1% penicillin-streptomycin (Lonza Walkersville), and recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech). For flow cytometry analysis of myeloid cells, we used FITC-conjugated anti-Gr1 (BD PharMingen), FITC-conjugated anti-Ly6G (BD PharMingen), PerCP-conjugated anti-CD11b (BioLegend), APC-conjugated anti-Ly6C (BioLegend), APC-conjugated anti-CD11c (eBioscience), and PE-conjugated anti-F4/80 (eBioscience). For the flow cytometry analysis of lymphocytes, we used FITC-conjugated anti-CD4 (eBioscience), APC-conjugated anti-Rorγt (eBioscience), PE-conjugated anti-Foxp3 (eBioscience), and 7-AAD Viability Staining Solution (eBioscience). For the Western blotting analysis, anti-phospho-p70 S6 kinase (Thr389) (Cell Signaling, #97596 S), anti-p70 S6 kinase (Cell Signaling, #9202), anti-phospho-Akt (Ser473) (Cell Signaling, #4060), anti-Akt (Cell Signaling, #2920), and anti-β-actin antibodies (Sigma-Aldrich) were purchased.

LP cells from colon or splenic immune cells were sorted using a FACS flow cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo_V10 (Treestar, Ashlan, OR). The gating strategy has been shown in supplementary Figure 8. For macrophage and neutrophil analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD11b (11-0112-82, eBioscience, San Diego, CA), APC-conjugated anti-CD11c (17-0114-81, eBioscience), PE-cyanine7-conjugated anti-F4/80 (25-4801-82, eBioscience), PE-conjugated anti-CD206 (12-2061-80, eBioscience), and PE-conjugated anti-Ly6G (12-5931-81, eBioscience); for T cell analyses, cells were incubated with the following antibodies: APC-conjugated anti-CD3 (17-0032-82, eBioscience), FITC-conjugated anti-CD4 (11-0042-81, eBioscience), and PE-conjugated anti-CD8 (12-0081-81, eBioscience); for B cell and NK cell analyses, cells were incubated with the following antibodies: FITC-conjugated anti-CD19 (11-0193-81, eBioscience), and PE-conjugated anti-NK1.1 (12-5941-81, eBioscience).

Astragalin was modified into astragalin-galactoside (Ast-Gal) using β-galactosidase from Bacillus circulans, and purified by the medium pressure chromatography with silica C-18 column followed by Sephadex LH-20 column [25 (link)]. Ast-Gal was identified by nuclear magnetic resonance to be kaempferol-3-O-β-d-glucopyranosyl-(1->6)-β-d-galactopyranosyl-(1->4)-β-d-galactopyranoside. The water solubility of Ast and Ast-Gal were 28.2 ± 1.2 mg/L and 38,800 ± 2.8 mg/L, respectively. Anti-CD3ε, anti-CD28, and anti-IL-12p70 antibodies were purchased from BD Biosciences (San Diego, CA, USA). Mouse recombinant IFN-γ and IL-4 were purchased from PROSPEC (East Brunswick, NJ, USA). Mouse recombinant IL-6 and TGF-β were purchased from PEPROTECH (Rocky Hill, NJ, USA). FTIC-conjugated anti-CD4, FTIC-conjugated anti-MHC Class II, FTIC-conjugated anti-IgG 2b/k, PE-conjugated anti-IFN-γ, PE-conjugated anti-IL-4, PE-conjugated anti-CD80, PE-conjugated anti-CD86, PerCP-Cy5.5-conjugated anti-CD4, and APC-conjugated anti-IFN-γ antibodies were purchased from BD Biosciences. PE-conjugated anti-Rat IgG2a, PE-conjugated anti-IL-13, APC-conjugated anti-IL-17A, and APC-conjugated anti-CD11c were purchased from eBioscience (San Diego, CA, USA).

Splenocytes and bone marrow cells from infected mice were analysed by flow cytometry. Cells were stained with PE-conjugated anti-DNGR-1, APC-conjugated anti-CD11c, FITC-conjugated anti-MHCII and CD4, Pacific blue-conjugated anti-CD8, and Percp-conjugated anti-CD3 (all obtained from eBioscience), as previously described^{13 (link)}. For intracellular staining, cells were stimulated with BMDC pre-incubated with fixed parasite. 2 hours later, Brefeldin A was added and cells were incubated for further 4 hours. After fixation, cells were permeabilised and stained with APC-conjugated anti-INFγ. Flow cytometric analysis was performed with a BD LSRFortessa™ cell analyser (Becton Dickinson). 5.10⁵ cells per sample were acquired and analysed with the FACSDiva or with the flowjo software.