Comprehensive Evaluation of Influenza Pathogenesis

Mouse lungs were lavaged with 1 mL sterile PBS for inflammatory cell counts. Cytospin preparations of BAL fluid were stained using Diff-Quick (Sigma St. Louis, MO) and differential cell counts were conducted based on cell morphology. The superior lobe of the right lung was homogenized in sterile PBS (1ml) by mechanical grinding. The lung homogenate was use for bacterial colony counting cytokine analysis by Bio-plex Multiplex immunoassay (BioRad, Hercules, CA). Middle and inferior lobes of the right lung were snap-frozen and homogenized under liquid nitrogen for RNA extraction using an RNA isolation kit (Agilent Technologies, Santa Clara, CA). RNA analysis was performed by RT-PCR using Assay on Demand TaqMan probes and primers (Applied Biosystems, Foster City, CA). For bacterial dissemination studies, the liver, spleen, and kidneys were homogenized in PBS and plated for colony counts. Influenza burden was determined by RT-PCR for matrix protein expression as previously described^{56 (link)}.