Cd25 fitc

For flow cytometry, the following fluorochrome-conjugated monoclonal antibodies were used. BD Biosciences: HLA-DR-APC (Clone: G46-6), CD86-FITC (Clone: FUN-1), CD80-PE (Clone: L307.4), CD54-APC (Clone: HA58), CD25-FITC (Clone: M-A251), CD127-BV421 (clone HIL-7R-M21), IFN-γ-FITC (Clone: 4S.B3), IL-4-PE (Clone: MP4-25D2); eBioscience: FoxP3-APC (Clone: 236A/E7), IL-17A-PE (Clone: Ebio64cap17); Beckman Coulter: CD40-PE (Clone: MAB89); Biolegend: CD4-PerCP (Clone: SK3). Cell viability was detected using the fixable viability dye eFluor 506 (eBioscience).
Antigen affinity-purified polyclonal anti-human TLR4 goat IgG was purchased from R&D systems. Cytokines (recombinant human granulocyte-macrophage colony-stimulating factor and IL-4), MicroBeads (CD14 and CD4) and cell purification units were obtained from Miltenyi Biotec. Protein-A agarose beads were from Cell Signalling Technology, and E. coli 055:B5 LPS and Polymyxin B-conjugated agarose beads were from Sigma-Aldrich. TLR4 signaling inhibitor CLI-095 and CpG ODN 2006 were procured from InvivoGen.

The cells were divided into several groups and then stained with fluorophore-conjugated antibodies, including CD11b-FITC or BV421 (eBioscience), Gr-1-PerCP Cy5.5 or APC (eBioscience), CCR5-APC or PE (BioLegend, CA, USA), CD45-AmCyan (BioLegend), CD4-PE (eBioscience), and CD25-FITC (eBioscience) for 30 min at 4°C in staining buffer (PBS with 10% FBS). CCL5-PE/Cyanine7 (BioLegend), Foxp3-APC (eBioscience), and interferon-gamma (IFN-γ)-FITC (eBioscience) were used for intracellular staining after culturing with fixation/permeabilization medium (eBioscience). Data were acquired through FACS AriaIII (BD Biosciences) and analyzed with FlowJo X (BD Biosciences).

Cells were characterized by flow cytometry using the monoclonal antibodies (mAbs) CD11b/c-APC, CD80-PE, CD86-PE, MHCI-FITC, and MHCII-PE (BioLegend, San Diego, CA, USA). For analysis of the glycocalyx, lectins from Maackia amurensis (MAL II, indicating α2-3 Sia) and Sambucus nigra (SNA-I, indicating α2-6 Sia) were used (Vector Labs). Lectins were biotin conjugated. PE-streptavidin was used for detection. Negative controls for non-specific fluorescence were used, these consisted of PE-streptavidin staining solutions in the absence of the lectin conjugated to biotin. Lectins were prepared in lectin staining buffer (PBS containing 1% FBS, 1 mmol/L CaCl₂, and 2 mmol/L MgCl₂) and resuspended in FACS buffer (PBS containing 2% fetal calf serum and 0.01% NaN₃, all from Sigma-Aldrich) before analysis using a FACS Canto II (BD Biosciences, Oxford, UK).
For analysis of the assays involving lymphocytes from the lymph nodes and spleen, the following mAbs were used CD3/PE, CD8/PE-Cy7, CD4/APC (BioLegend), and CD25/FITC (eBioscience, San Diego, CA, USA). Prior to staining, cells were washed with FACS buffer. mAbs were diluted in 50 µL FACS buffer, added to the cells, and incubated for 15 min at 4°C. To remove any unbound antibodies, the cells were washed three times with FACS buffer. The cells were then filtered through a nylon mesh (40 µm) before analysis in the cytometer.