Cd40 percp cy5

To investigate if SPION^Dex inhibit DC maturation, iDCs were incubated in the presence of SPION^Dex. Therefore, SPION^Dex at iron concentrations of 50, 100 and 200 µg/mL were incubated with iDCs at a cell density of 10⁶/100 µL in 6 well plates at 37 °C for 48 h. The DC medium containing IL-1β (200 U/mL), IL-6 (1000 µ/mL), tumor necrosis factor (TNF)-α (10 ng/mL, all from ImmunoTools, Friesoythe, Germany) and prostaglandin E2 (PGE2) (1 µg/mL, Pfizer, New York, USA) served as positive control, pure DC medium served as negative control. After 48 h of incubation, the matured DCs (mDCs) were harvested from the 6 well plates by washing with ice-cold PBS-EDTA. The cells were centrifuged at 200 rcf for 12 min, the supernatant removed and the cell number adjusted to 1 × 10⁶/mL with PBS containing 2 vol% fetal calf serum (FCS). To analyze the expression of surface maturation markers, 50 µL cells were stained with fluorescence labelled antibodies (CD83-PE/Cy7, CD40-PerCP/Cy5.5, CD80-APC, CD86-PerCP/Cy5.5 (all from BioLegend, San Diego, CA, USA) in PBS containing 2% FCS for 30 min. Isotype antibodies were used as controls. Subsequently, cells were washed and fixated with 100 µL PBS containing 1% paraformaldehyde. Then, cells were analyzed in flow cytometry.

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).