Cross-Species Genome-Wide Analysis Reveals Molecular and Functional Diversity of the Unconventional Interferon-? Subtype

Shields, Lauren E.; Jennings, Jordan; Liu, Qinfang; Lee, Jinhwa; Ma, Wenjun; Blecha, Frank; Miller, Laura C.; Sang, Yongming

Blood (∼20 ml/pig) was collected by jugular venipuncture from anesthetized pigs. Immediately after euthanasia, cubes of ∼0.5 cm 3 were dissected from lung or other indicated tissues (9, 21, 22) . Lungs were lavaged with 300 ml/each of 10 mM PBS (pH 7.4) (9, 13) . Samples were placed on ice and peripheral blood mononuclear cells (PBMCs) and macrophages (Mφs) were isolated from the heparinized blood samples and lavage fluids, respectively, within 4 h after collection. PBMCs were isolated using a 60% Ficoll-Paque Plus gradient (GE Healthcare, Piscataway, NJ). Monocytes were isolated from the PBMCs with an anti-CD14 antibody (Ab), then the CD14 − population was used to isolate cDCs with a CD172 antibody (9, 13, 22) , using magnetic beads conjugated with the corresponding secondary Ab (Miltenyi Biotec, Auburn, CA). The PRRSV-permissive mDCs were generated by culturing monocytes in the presence of IL-4 and GM-CSF for 7 days as described (9, 13, 22) . Lavage fluid was centrifuged at 400× g for 15 min to collect cells and further isolate Mφs by plastic adherence (9, 22) . Cells were used immediately or cryopreserved in Recovery TM cell culture freezing medium (Invitrogen, Carlsbad, CA).