Mouse il 23 quantikine elisa kit

CD11c⁺ DCs from lymph nodes were selected with anti-CD11c beads according to the protocol of CD11 MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured in a Roswell Park Memorial Institute (RPMI) medium 1640 with 10% fetal bovine serum. DCs (1.0 × 10⁵ cells per 200 μL well) were stimulated with 1 μg/mL of R848 (InvivoGen, San Diego, CA, USA) for 24 h and the total RNA was then isolated using Trizol (Invitrogen, Waltham, MA, USA). Then, the mRNA expression levels were determined as mentioned above. The supernatants were also harvested and the IL-23 and IL-10 protein levels were quantified using a mouse IL-23 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and a mouse IL-10 Quantikine ELISA Kit (R&D Systems), according to the manufacturer’s instructions. In some experiments, the DCs were stimulated with 1 μg/mL of R848 for 15 min or 1 h and the phosphorylated NF-κB p65 expression in DCs was analyzed with a NFκB p65 ELISA kit (Enzo Life Sciences, Tokyo, Japan) according to the manufacturer’s instructions. These assays employed the quantitative sandwich enzyme immunoassay technique. Optical densities were measured at 450 nm using a Bio-Rad Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).