Type 1 Interferon Induction of Natural Killer Cell Gamma Interferon Production for Defense during Lymphocytic Choriomeningitis Virus Infection

Mack, Ethan A.; Kallal, Lara E.; Demers, Delia A.; Biron, Christine A.

Flow cytometric analysis. Detection of surface markers, intracellular IFN-␥, and intracellular total STAT1 and STAT4 was done with minor modifications as previously described (9, 46) . Briefly, PECs were incubated with 2.4G2 antibody (BioXCell, West Lebanon, NH) to block nonspecific binding to the Fc receptor. Cell surface staining was then performed with antibodies specific for the following: TCR-␤-FITC, TCR-␤peridinin chlorophyll protein (PerCP), NK1.1-phycoerythrin (PE) (all from eBioscience, San Diego, CA), TCR-␦-FITC, and CD45.1-FITC (BD Biosciences, Franklin Lakes, NJ). For cell surface staining only, cells were fixed with 2% paraformaldehyde. For intracellular staining of IFN-␥, cells were fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and then stained with IFN-␥-allophycocyanin (APC) (BD Biosciences). For intracellular staining of total STAT1 and STAT4, cells were fixed and permeabilized with Cytofix/Cytoperm and then further fixed and permeabilized with ice-cold pure methanol. Subsequent staining was performed with combinations of STAT1-PE and STAT4-APC (custom prepared by BD Biosciences). The following isotype controls were included in each experiment: mouse IgG2a, mouse IgG2b, mouse IgG1, rat IgG1, rat IgG2a, rat IgG2b, and hamster IgG (BD Biosciences or eBioscience). Samples were acquired using a FACSCalibur (BD Biosciences) with the Cell-Quest Pro software package (BD Biosciences). Laser outputs were 15 mW at 488-and 635-nm wavelengths. At least 120,000 events were collected within a live-cell gate that was determined by forward and side scatter.