Facsaira

To detect the effect of DAC on the DC surface markers, DAC-treated and -untreated DCs were stained by anti-human CD86-APC, CD80-PE, HLA-DR-PE/Cy5, CD83-PE and CD40-PerCP-Cy5.5 (all antibodies were from BioLegend, San Diego, USA) at 4°C for 30 minutes. The expression of cell surface markers was detected by flow cytometry (FACSAira, BD Biosciences, Franklin Lakes, USA) and analyzed as the median fluorescence intensity (MFI). The histograms with overlays were made by FlowJo software (Treestar, Inc., San Carlos, USA).
To investigate whether DAC affects the function of DCs in relation to its effect on differentiating T cells into Th1/Th17 subsets, CD4⁺ cells were co-cultured with DAC-treated or -untreated DCs for 5 days as previously described [11 (link)]. Then 100 ng/ml PMA (Sigma-Aldrich, St. Louis, USA) and 1 μg/ml ionomycin (Sigma-Aldrich, St. Louis, USA) were added to the cells at 37°C for 1 hour and subsequently incubated for an additional 4 hours with 10 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, USA). Anti- IFN-γ-FITC and IL-17A-PE antibodies (both from eBioscience, San Diego, USA) were used to perform the intracellular staining for 30 minutes at 4°C. The frequencies of CD4⁺ IFN-γ⁺ and CD4⁺ IL-17A⁺ cells were detected with the FACSAira flow cytometer (BD Biosciences, Franklin Lakes, USA).

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.