Cd83 fitc

MoDC were exposed to pI:C (25 µg/ml), CL307 (1 µg/ml), R848 (1 µg/ml) LPS (100 ng/ml), or pI:C/LyoVec (1 µg/ml) (Invivogen) with or without the addition of ssON, and assessed 48 h later by flow cytometry using monoclonal antibodies (Abs) targeting CD1a and the moDC maturation markers CD86, CD83, and CD80 (CD1a-Bv510, CD86-APC, CD83-FITC, CD80-PE; all from BD Biosciences). Where chloroquine was used, cells were pre-incubated with 10 µM chloroquine for 1 h at +37 °C. Dead cells were excluded using Live/Dead fixable near-IR dead cell stain kit (Life Technologies).

Lipofectamine® 2000 Transfection Reagent and TREM-1 siRNA were purchased from Invitrogen (Carlsbad, USA). TRIzol, LPS, and human LDL were purchased from Sigma-Aldrich (St. Louis, USA). LP17 (LQVTDSGLYRCVIYHPP) and a sequence-scrambled control-peptide (TDSRCVIGLYHPPLQVY) were produced by GL Biochem (Shanghai) LTD, as described by Gibot et al. [18 (link)]. The peptides were obtained with good yields (>99%), purified, and confirmed by preparative chromatography. These peptides were free of endotoxin. The anti-CD14 and CD4 magnetic beads were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). The ELISA kits, CD1a-FITC, CD40-FITC, CD86-FITC, CD83-FITC, CD14-FITC, and HLA-DR-PE antibodies were purchased from BD (Franklin Lakes, USA). The anti-TREM-1 primary antibody and Human sTREM-1 ELISA kit were purchased from R&D (HK, China). The anti-SOCS1and GAPDH primary antibodies were purchased from Cell Signaling (Denver, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). Recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) were purchased from R&D Systems (Minneapolis, USA).

The culture media used were RPMI-1640, IMDM, McCoy’s 5a, DMEM, or Ham’s F-12. These media were supplemented with 2 mM L-glutamine, 20 mM HEPES, 1% antibiotic-antimycotic solution (all obtained from Invitrogen, Carlsbad, CA, USA), and 10% heat-inactivated fetal bovine serum (FBS) (HyClone, Logan, UT, USA). Recombinant human GM-CSF, IL-4, TFF2, IL-8, and MIP-3β were obtained from Peprotech (Peprotech, Rocky Hill, NJ, USA). LPS was from Sigma Chemical Co. (St. Louis, MO, USA). The following fluorochrome-labeled monoclonal antibodies were used to analyze phenotypes of cells in peripheral blood mononuclear cells (PBMC) or cultured DC: CD1a-PE, CD40-FITC, CD80-PE, CD83-FITC, CD86-PE, and HLA-DR-FITC (all from BD-Pharmingen, San Jose, CA, USA).

Cell phenotype was assessed by flow cytometry (BD LSRFortessa; BD Biosciences) after incubation with the following fluorescein-labeled antibodies: CD45-allophycocyanin (APC)-eFluor780, CD3e-fluorescein isothiocyanate (FITC), CD4-APC, CD8a-phycoerythrin (PE)-cy7, CD25-PE-cy7, Foxp3-PE, IFN-γ-AlexaFluor488, IL-4-PE-cy7, CD107a-PE, granzymeB-peridinin chlorophyll (PerCP)-eFluor710, perforin-FITC, CD11c-PE-cy7, CD80-PerCP-eFluor710, CD83-FITC, B7-H4-PE, and CD31-PE. All antibodies were purchased from eBioscience (San Diego, CA, USA), except CD4-APC, CD8a-PE-cy7, and Foxp3-PE (BD Biosciences). Intracellular antigens were determined after incubation with ionomycin (500 ng/mL; Abcam, Cambridge, UK) and phorbol-12-myristate13-acetate (10 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) for 1 h and monensin (2 μM; eBioscience) for an additional 4 h. Fixation and permeabilization were performed prior to antibody incubation, and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

For THP-1 activation assays: anti-mouse TLR2 (mab2-mtlr2) and anti-human TLR4 (mabg-htlr4) antibodies were purchased from InvivoGen. For flow cytometry: CD83 FITC (BD #556910), CD80 PE (BD #557227), CD86 APC (BD #555660), CD45 BV786 (BD #563716), HLA-ABC Pe-Cy7 (BD #561349), HLA-DR BV421 (BD #564244), and CD11c APC (BD #559877) were purchased from BD Biosciences. To determine cell viability, LIVE/DEAD Fixable Yellow Stain (LDY #L34968, Invitrogen, Waltham, MA, USA) was used.