Anti human cd40 pe

For the detection of surface markers and intracellular proteins, the cells were harvested after trypsin treatment and resuspended in ice-cold PBS. The following antibodies (Abs) conjugated with Annexin V-Fluorescein isothiocyanate (FITC) or PE were purchased from eBioscience (San Diego, CA, USA): anti-human CD11b FITC (11–0118), anti-human CD11b APC (17–0118), anti-human CD14 FITC (11–0149), anti-human CD40 PE (12–0401), anti-human CD68 PE (12–0689), and anti-mouse major histocompatibility complex (MHC) Class II (I-A) PE (12–5322). The cells were stained with the fluorochrome-conjugated Abs for 15 min and then washed twice with PBS containing 2% FCS. The stained cells were recorded using a FACS Calibur platform (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Cell culture medium for PBMCs and DCs was RPMI 1640 (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS (Gibco, Carlsbad, CA, USA) and 100 U/mL streptomycin/penicillin. Anti-human CD3, anti-human CD28, anti-human CD4-FITC, anti-human CD25-APC, anti-human Foxp3-PE, anti-human IFN-γ-PE, anti-human IL-17-PE, anti-human CD11c-APC/Cy7, anti-human HLA-DR-PerCp/Cy5.5, anti-human CD40-PE, and anti-human CD86-PE were all from eBioscience, San Diego, CA, USA. TRIzol, PrimeScript RT reagent kit, and SYBR Green Master Mix were from Takara Biotechnology, Dalian, China. Recombinant human GM-CSF and recombinant human IL-4 were obtained from PeproTech, Rocky Hill, NJ, USA. Meanwhile, human magnetic CD14 isolation kit and CD4 isolation kit were bought from Miltenyi Biotec, Auburn, CA, USA. Lipopolysaccharides (LPS) were from Sigma-Aldrich, St Louis, MO, USA, and recombinant human IL-37 was bought from AdipoGen AG, Liestal, Switzerland. Oxidised low density lipoprotein (oxLDL) was acquired from Yiyuan, Wuhan, China, and phorbol myristate acetate, ionomycin, and monensin were from Alexis Biochemicals, San Diego, CA, USA.