Anti human il 17a pe

For IL-17 and IFN-γ staining, cultured CD4+T cells were stimulated with PMA (50ng/mL) and ionomycin (1g/mL) for 1 h at 37°C, and brefeldin A (Sigma-Aldrich) for another 4 h, then washed, fixed and permeabilized. Fluorescent anti-human CD3-APC, anti-human IL-17A-PE, anti-human IFN-γ-PE-cy7, and anti-human IgG isotype were purchased from eBiosciences.

DAC-treated and –untreated DCs were incubated with anti-human HLA-DR- phycoerythrin-cyanin 5, CD86-allophycocyanin, CD80-phycoerythrin, CD40-peridinin chlorophyll-cyanin 5.5 and CD83-phycoerythrin at 4 °C for 30 minutes. All these antibodies were bought from BioLegend (CA, USA). Flow cytometry (FACSAira, BD Biosciences, USA) tests were performed to measure the changes of the markers. The results were shown as median fluorescence intensity (MFI) and were analyzed by FlowJo software (Treestar, Inc., CA, USA). For the co-culture experiment, CD4⁺ cells co-cultured with DAC-treated or –untreated DCs were incubated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μg/ml ionomycin for 1 hour at 37 °C and then added 10 μg/ml brefeldin A (all from Sigma-Aldrich, MO, USA) for an additional 4 hours. The frequency of CD4⁺ IFN-γ⁺ and CD4⁺ IL-17A⁺ cells were detected by flow cytometry. Briefly, CD4⁺ cells were permeabilized by using the Intracellular Fixation & Permeabilization Set (eBioscience, CA, USA). The cells were subsequently stained with anti-human IL-17A-PE (eBioscience, CA, USA) and anti-human IFN-γ-FITC (eBioscience, CA, USA) for 30 minutes at 4 °C.