Anti il 12 p40

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).