Anti cd83

To analyze DC surface markers, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF for seven days. DCs were treated with 100 ng/mL LPS for 24 h in the absence or presence of 10 mM D-2-HG or L-2-HG. Cells were harvested and stained with anti-CD1a & anti-HLA-DR (Beckman Coulter, Krefeld, Germany), anti-CD80 (Biolegend, San Diego, CA, USA), anti-CD83 (eBioscience, San Diego, CA, USA), and anti-CD86 (BD Bioscience, Franklin Lakes, NY, USA) at 4 °C for 30 min. After washing, DCs were resuspended in FACS wash buffer. Flow cytometric measurement was performed using a BD FACS Calibur instrument (BD Bioscience).
For intracellular detection of IL-12 p35 and IL-12 p40, monocytes were cultured in RPMI medium supplemented with IL-4 and GM-CSF. After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStop^TM, BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/Cytoperm^TM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls. Cells were analyzed using a BD FACS Calibur instrument (BD Bioscience).

Expression of CD90, CD105, CD45, and CD11b/c (eBioscience, USA) on MSCs surface was assessed by flow cytometry (Becton Dickinson, USA). The expression of CD11b/c, CD80, CD83, CD86, and MHC II (eBioscience, USA) on DCs surface was identified using flow cytometry. Each group of DCs were collected on the sixth day. After washing with PBS, the cells were immunolabeled with monoclonal anti-CD80, anti-CD83, anti-CD86, and anti-MHC II (eBioscience, USA) antibodies, as well as their isotype control antibodies. After that they were incubated in darkness at 4°C for 30 min and were detected using FACSCalibur flow cytometer.

Immature DCs (10⁷) were stimulated for 6 h with LPS (1 µg/ml E. coli strain O111:B4, Calbiotech Merck) or LPS in combination with IFN-γ (0.02 µg/ml, BD Pharmingen) with or without Dex (10⁻⁸ M, Sigma-Aldrich) for 20 min before adding other reagents. To monitor maturation, DCs (10⁵) were stained with 5 µl of mix including anti-MHC-I, anti-MHC-II, anti-CD11b, anti-CD11c, anti-CD80, anti-CD83 and anti-CD86 (all from eBioscience, Austria).
T-cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-Vα2 TCR for OT-I and OT-II mice (eBioscience). Cell viability was analysed with DAPI (Sigma-Aldrich). Apoptosis was measured with Annexin V (BD Pharmingen). CFSE (7 µM, Invitrogen) or Cell Proliferation Dye eFluor 670 (CPD, 5 µM, eBioscience) were used to detect proliferation. Flow cytometry was done on an LSR II (BD Pharmingen). Data were analysed by FlowJo (Version 9.6.2 Treestar). The difference in apoptosis induction was calculated using absolute cell number, determined with BD Trucount tubes.
This method is based on lyophilized pellet, containing a known number of fluorescent beads, which dissolves once the monoclonal antibody reagent is added. Absolute numbers (cells/µl) of positive cells in the sample are calculated following the equation: number of cell events/number of bead events x Trucount bead concentration.