Hla dr pe cy5

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).

MSCs exposed to 0, 1.0, 1.5 mM R-2HG were determined by flow cytometry assay. A total of 5 × 10⁵ cells from single-cell suspensions were incubated for 30 min at room temperature with fluorochrome-conjugated monocolonal antibodies against CD34-PE, CD73-APC, CD90-FITC, CD105-PE (eBioscience, San Diego, CA, USA), CD45-FITC, and HLA-DR-PE-Cy5 (Biolegend, San Diego, CA, USA). After washing with PBS, immunofluorescence analysis was performed by flow cytometry using a FACS Calibur system (Beckman Coulter, Miami, FL, USA) and data were calculated using the FlowJo Software. Appropriate isotype-matched antibodies were used as controls.