UNC93B1 Mediates Innate Inflammation and Antiviral Defense in the Liver during Acute Murine Cytomegalovirus Infection

Crane, Meredith J.; Gaddi, Pamela J.; Salazar-Mather, Thais P.

The following fluorochrome-conjugated mAbs were used in flow cytometric analyses: NK1.1-PE and TCRb-APC to distinguish NK cells; CD8a-PECy7 and TCRb-FITC to distinguish CD8+ T cells; and PDCA-1-APC (Miltenyi Biotec) to identify pDCs. Prior to surface staining, cells were incubated with anti-CD16/CD32 mAb to block nonspecific binding of Abs to Fcc III/ II receptors (clone 2.4G2). Unless otherwise noted, antibodies were obtained from BD Biosciences or eBioscience. For intracellular staining of cytokines, cells were treated with Brefeldin A (eBioscience) for 4 hours at 37uC, 5% CO 2 and permeabilized prior to labeling with IFN-a-FITC (R&D Systems), IFN-c-PE, or TNF-a-APC (BD Biosciences). When indicated, leukocytes were stimulated for 5 hours with 100 ng/mL MCMV M45 peptide in addition to Brefeldin A treatment. Samples were acquired using a FACSCalibur and analyzed with BD Cell Quest software. For analysis, viable cells were gated by FSC and SSC. Isotype controls were used to set positive analysis gates.