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.

Sample preparation. PECs were extracted on the indicated days of infection. LCMV-infected and control uninfected (0) mice were anesthetized and bled retro-orbitally to collect serum samples. Mice were humanely sacrificed, by cervical dislocation after anesthetization, and the peritoneal cavity was lavaged with 5 ml of cold serum-free RPMI medium (Gibco, Invitrogen, Carlsbad, CA). Mice were gently agitated to increase PEC suspension in lavage medium. For analysis of surface marker expression or total STAT levels, PECs were resuspended in staining buffer containing 2% heat-inactivated fetal bovine serum (FBS) and then prepared for flow cytometric analysis as described below. For examination of intracellular IFN-␥, PECs were resuspended in complete media containing 5 g/ml brefeldin A (Sigma, St. Louis, MO), without additional stimulation, for 4 h at 37°C. Cells were then stained as described below. Peritoneal lavage fluids and serum samples were aliquoted and stored at Ϫ80°C for later use in cytokine and viral titer measurements.