Anti human il 4 pe

Monocyte-derived DCs and macrophages were counted, washed, and then co-cultured with allogeneic peripheral blood lymphocytes (PBL) for three, five, or nine days in RPMI-1640 medium (Sigma-Aldrich) at a moDC/macrophage: T-cell ratio of 1: 10 at 37 °C. To determine which T-lymphocyte populations were polarized by the preconditioned DCs and macrophages after three, five or nine days the T cells were stimulated with 1 µg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 h, and the vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) 12 h before the cell staining. The cells were labeled with anti-human CD4-Peridinin Chlorophyll Protein Complex (PerCP), CD8-PE and/or anti-human CD25-PE-conjugated antibodies (BioLegend). Following this, they were fixed and permeabilized by using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend), and anti-human FoxP3-APC (R&D System) antibodies. Fluorescence intensities were measured by FACS Calibur cytometer (BD Biosciences) and data were analyzed by the FlowJo v X.0.7 software (Tree Star).

Supernatants of moDCs and TU-CM-moDCs were harvested four days after monocyte separation or 24 hours after the stimulation of moDCs. The concentration of IL-6, IL-10, TNFα cytokines, and chemokine IL-8 was measured and validated using OptEIA kits (BD Biosciences) following the manufacturer’s instructions.
To determine which T-lymphocyte populations are responsible for the cytokine production, the T cells were stimulated with 1 μg/ml ionomycin and 20 ng/ml phorbol-myristic acetate (PMA) for 4 hours. The vesicular transport was inhibited by BD GolgiStop™ protein transport inhibitor (BD Biosciences) after the activation. The cells were then labeled with anti-human CD3-FITC, anti-human CD8-PE or CD4-PerCP, and anti-human CD25-PE antibodies (all from BioLegend). The samples were then fixed and permeabilized by BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit (BD Biosciences) and labeled again with anti-human CD8-PE or CD4-PerCP as well as with anti-human IFNγ-APC (BD Biosciences), anti-human IL-4-PE (R&D Systems), anti-human IL-10-Alexa Fluor 488, anti-human IL-17-PE (BioLegend). T cell staining panel is shown in S1 Fig. Fluorescence intensities were measured by Novocyte2000R Flow Cytometer (Agilent (Acea) Biosciences Inc., USA), and data were analyzed by the FlowJo v X.0.7 software (Tree Star).