Mouse il 12p40 elisa kit

Human PBMCs obtained from healthy donors who provided informed consent were purchased from HemaCare. Human PBMCs (1 × 10⁶) cultured in 96 wells plate were stimulated in triplicate for 24 h with K3, K3-SPG, and D35 at the concentrations indicated in the figure legends. Culture supernatants were harvested and subjected to assays with human IFN-α ELISA kit (Abcam) and human IL-12p40 ELISA kit (BioLegend), according to the manufacturer’s instructions. The experiments were approved by the Ethics Committee of Kyoto University Graduate School of Medicine (R1004). To prepare mouse splenocytes, spleens were harvested from sacrificed mice and mechanically homogenized into single-cell suspensions filtered through 40-μm nylon filters into PBS with additional red blood cell (RBC) lysis using RBC Lysing Buffer Hybri-Max (Sigma-Aldrich). Mouse splenocytes (1 × 10⁶) cultured in 96 wells plate were stimulated in triplicate for 24 h with K3 and K3-SPG at the concentrations indicated in the figure legends. Culture supernatants were harvested and subjected to assays with mouse IFN-α ELISA kit (PBL Assay Science) and mouse IL-12p40 ELISA kit (R&D Systems) according to the manufacturer’s instructions.