Anti il 12 p35

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).

Spleen were removed from mice and gently meshed in DMEM containing 10% FBS (Life Technologies) to prepare for single cell suspensions. CD4⁺/CD25⁻ T cells were isolated by the CD4⁺CD25⁻ T cell isolation kit (Miltenyi Biotec) according to manufacturer's instruction. The purity of CD4⁺CD25⁻ populations was around 90%. CD11c⁺ DCs isolated from colitic mice receiving the different treatments were cultured for 24 hrs in the presence or absence of GTS-21 (a specific α7nAChR agonist, 100 µM) before medium was washed and co-cultured with CD4⁺CD25⁻ T cell isolated from naïve mice at ratio of 1∶3 (DCs:T cells) [25] (link) in plates coated with 10 µg/ml⁻¹ of anti-CD3 and 2 µg/ml⁻¹ of anti-CD28. In neutralization experiments, these cultures were treated with 10 µg/ml anti-IL-12p35 or anti-IL-23p19 (R&D Systems) neutralizing mAb to block the potential activities of endogenous sources of these cytokines. In a separated set, recombinant (r) IL-12p70 or rIL-23 protein (25 ng/ml⁻¹; R&D Systems) were added to the cell culture medium. Cell culture supernatants were collected at 24 hrs, and interferon-gamma (IFN-γ), IL-4 and IL-17 levels were analyzed by ELISA (R&D Systems).