Recombinant mouse ifnβ standard

For cytokine measurement in culture supernatants, spleen or BM cells from individual mice in the same group were pooled prior to FACS purification. FACS-purified pDCs (3 × 10⁴ cells/well) were incubated in 120ul RPMI-1640 supplemented with 10% (vol/vol) fetal bovine serum, L-glutamine, penicillin-streptomycin, and HEPES pH 7.2) supplemented with 50μM β-mercaptoethanol in the presence or absence of CpG-B 1668 (Integrated DNA Technologies) at 0.1μM (Flt3L culture-derived pDC) or 1μM (splenic and BM pDC) or CpG-A 1585 (InvivoGen) at 0.5uM for 6 or 15 hours at 37°C. Supernatants were collected and stored at −80°C until further analysis. IFN-I bioactivity was measured with reference to a recombinant mouse IFNβ standard (Sigma-Aldrich) using ISRE-L929 reporter cells (L-929 cell line transfected with an interferon-sensitive luciferase) (Jiang et al., 2005 (link)). IFNα and IFNβ were measured by Lumikine mIFNα and Lumikine Xpress mIFNβ ELISA (InvivoGen), respectively, following manufacturer instructions. TNFα was measured by Mouse TNF alpha ELISA Ready-SET-Go!® (eBioscience) following manufacturer instructions. Graphs depicting cytokine measurements in this paper represent individual wells of stimulation.