Anti human hla dr percp cy5

Fluorescent immune cell probes for the delineation of monocyte subsets included anti-human CD14-V450, anti-human CD16-FITC, anti-human HLA-DR-PerCP-Cy5.5, anti-human CD33-APC, anti-human CD11b-PE, and IgG isotype controls (ebioscience or BD Biosciences). Cells were counted using an LSRII flow cytometer with the data was analyzed with BD FACSDIVA software. The submitted flow cytometry gating paradigm (Additional file 2: Figure S2) depicts cell populations for analysis of mature monocyte populations and the immature MDSC population.

Isolated pan monocytes were incubated with Fc blocker to avoid non-specific binding. Both isolated monocytes and fresh blood samples were then incubated with anti-human CD14-V450 antibody (eBioscience, San Diego, CA, USA), anti-human CD16-FITC (eBioscience, San Diego, CA, USA), anti-human HLA-DR-PerCP Cy5.5 (eBioscience, San Diego, CA, USA), anti-human TIM3-PE (eBioscience, San Diego, CA, USA). One additional lysing step to remove red blood cells was done for peripheral blood samples. Dead cells were stained using LIVE/DEAD Fixable Blue Dead Cell Stain Kit (Molecular Probes, Eugene, OR, USA). The cells were immediately analyzed using an LSR II™ 13 color flow cytometer configured with 355, 488, 405, 561, and 633 lasers.

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.