Cultivation and Characterization of Macrophage and DC Lines

RAW 264.7 (RAW), NIH3T3, and 293T cells (American Type Culture Collection) were maintained in DMEM with 10% FBS or 10% donor serum (for NIH3T3). IRF8^−/− macrophage cell line, CL2 cells were cultured in RPMI 1640 and 10% FBS plus rM-CSF (5 ng/ml) (32 (link)). Bone marrow (BM)-derived macrophages were obtained by culturing BM mononuclear cells in the presence of 20 ng/ml M-CSF (Invitrogen) for 5–6 d. BM-derived IRF8^−/− DCs were cultured in the presence of Flt3L for ∼4 wk. These cells exhibited a DC progenitor-like property and differentiated into DCs after IRF8 transduction. Detailed procedures and the properties of these DCs will be presented elsewhere (P. Tailor, unpublished observations). Procedures for quantitative RT-PCR (qRT-PCR), in vivo SUMOylation, and coimmunoprecipitation assays were performed, as described (19 (link)).