Mouse Fibroblast Isolation and Immortalization

Primary mouse fibroblasts (mouse embryonic fibroblasts [MEF] and mouse newborn cells [MNC]) were isolated from C57BL/6 and BALB/c mouse embryos and newborns according to described protocols (65 (link)). Immortalized mouse fibroblasts were generated from primary C57BL/6 and BALB/c MEF by crisis immortalization (66 (link)). HeLa:IFNLR and 3T3-ISREluc:IFNLR cells were generated by transfection of HeLa cells (ATCC CCL-2) and NIH 3T3:ISREluc cells (11 (link)) with plasmids pcDNA-hIFNLR1 (subcloned from pUNO1-hIL28R; InvivoGen) and pUNO1-mIL28R (InvivoGen), respectively, and subsequent selection with the appropriate agent. All cells were grown in Dulbecco modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum, 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM glutamine. Cell culture media and supplements were obtained from Gibco/Life Technologies. Mouse IFN-α was purchased from PBL, mouse IFN-γ was purchased from Merck Millipore, human and mouse IFN-λ was purchased from R&D Systems, and ruxolitinib was purchased from Cell Guidance Systems.