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The extracted samples were evaporated, reconstituted in a small volume, and the eicosanoids were separated by reverse phase LC using a Synergy C18 column (Phenomenex). The eicosanoids were analyzed by tandem quadrupole MS (MDS SCIEX 4000 QTRAP, Applied Biosystems) operated in the negative-ionization mode via multiple-reaction monitoring (MRM) using transitions that were optimized for selectivity and sensitivity. Authentic standards were analyzed under identical conditions, and eicosanoid quantitation was achieved by the stable isotope dilution method and comparison with 141 quantitation standards. Data analysis was performed using the MultiQuant 2.1 software (Applied Biosystems). Selleck Thiazovivin A complete list of all metabolites included in the MRM profile is given in Table S2. The two-tailed Mann-Whitney test was used to determine statistical significance between population means (for mouse infections, red: X31sublethal; blue: PR8sublethal; black: PR8lethal. Magenta asterisks denote significance Laccase between PR8lethal and both X31sublethal and PR8sublethal infections; blue and red asterisks denote significance between PR8lethal and PR8sublethal or X31sublethal infections, respectively; and black asterisks denote significance between PR8sublethal and X31sublethal infections). Statistical significance: ?0.01?TGF-beta inhibitor TreeView. Raw files and processed data for lipidomics profilings are available at the Systems Influenza Website (http://www.systemsinfluenza.org) and at Metabolights (http://www.ebi.ac.uk/metabolights/; MTBLS31). Extended Experimental Procedures Mice were euthanized by CO2 inhalation. Broncho-alveolar lavage (BAL) was collected in 2?�� 1?ml washes with HBSS buffer. Cells from the BAL were collected by light centrifugation. Distinct cell types from the dissected lungs were isolated as described (Gereke et?al., 2007, Marsh et?al., 2009?and?Snelgrove et?al., 2008). Dispase digested lung tissue was disrupted into single-cell suspensions by passage through 100-��m, 70-��m, and 40-��m filters. Cells were incubated with anti-mouse CD16/32 (UCSF Monoclonal Antibody Core) and anti-mouse CD45 microbeads (Milteny) and separated by magnetic separation. RNA was extracted from distinct cell populations using TRIzol reagent (Invitrogen). Multiplex quantitation of chemokines and cytokines was conducted using the Luminex xMAP system with a MILLIPLEX MAP human cytokine/chemokine immunoassay panel or Mouse Cytokine/Chemokine panel as described by manufacturer (Millipore).
