Cd11c allophycocyanin

DMEM and RPMI 1640 cell culture media, antibiotics, and nonessential amino acids were purchased from Life Technologies (Grand Island, NY). CD14⁺ monocytes were isolated by MACS CD14 microbeads from Miltenyi Biotec (Auburn, CA). Human Abs, including CD16 allophycocyanin, CD16 FITC, CD14 FITC, CD40 FITC, and CD86 FITC, were purchased from eBioscience (San Diego, CA). Abs CD14 allophycocyanin, CD14 PE, CD163 PE, CD11c allophycocyanin, CD68 PE, CD206 allophycocyanin, DC-SIGN FITC, and isotype control Abs were purchased from BD Pharmingen (Franklin Lakes, NJ). Phospho-p44/42-ERK1/2 and anti-mouse IgG PE were obtained from Cell Signaling Technology (Danvers, MA). Human IL-10 Ab and mouse IgG1 isotype control were from R&D Systems (Minneapolis, MN). miR-27a inhibitor, mimic, and scrambled controls were purchased from Ambion Life Technologies (Carlsbad, CA). Lipofectamine RNAiMAX transfection reagent was from Life Technologies. The sprouty2 construct was obtained from OriGene (Rockville, MD), which was transfected by Roche (Indianapolis, IN) X-tremeGENE transfection reagent. ERK inhibitor, U0126, was procured from EMD Millipore (Billerica, MA).

PBMCs (1 × 10⁶) were Fc blocked (130-059-901; Miltenyi Biotec), stained, and acquired on a BD LSR II or a BD LSRFortessa (5 (link)). For intracellular cytokine staining, 1 × 10⁶ PBMCs were cultured in the presence of the appropriate antiegn (ColI or ColV) in DMEM containing 5% FBS at 37°C. After overnight incubation, the cells were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 5 h, with the addition of brefeldin A (420601; BioLegend) for the last 3 h. The following Abs were used: CD3-FITC or CD3–Alexa Fluor 700 (317306/100216; BioLegend or 557943; BD Biosciences), CD11b-BV421 (562632; BD Biosciences), CD56-allophycocyanin (555518; BD Biosciences), CD14–Alexa Fluor 700 (325614; BioLegend), CD16-allophycocyanin-H7 (560195; BD Biosciences), LAIR1-PE (550811; BD Biosciences), HLA-DR–PE-Cy7 (25-9956-41; eBioscience), CD123-BV421 (306017; BioLegend), lineage-BV510 (348807; BioLegend), CD11c-allophycocyanin (559877; BD Biosciences), PE-IgG₁ κ isotype control (55749; BD Biosciences), CD196/CCR6-FITC (11-1969-42; eBioscience), CD194/CCR4-PECy7 (561034; BD Biosciences), CD4-BUV395 (563552/563790; BD Biosciences), CD183/CXCR3-allophycocyanin (550967; BD Biosciences), IFN-γ-BV421 (563376; BD Biosciences), IL-17-PE (559502; BD Biosciences), γδ TCR-FITC (118105; BioLegend). Data were analyzed using FlowJo (Treestar).