Supernatants from disrupted lung were harvested during necropsy 5 wk post-M.tb infection and stored frozen at −80 °C. Samples were subsequently γ-irradiated on dry ice using a JL Shepherd Model 109–68 Cobalt-60 Research Irradiator (JL Shepherd & Associates, San Fernando, CA 91340) until 5 MRAD of exposure was reached. A non-diluted test sample was plated on 7H11 agar and incubated at 37 °C for >5 weeks to ensure sterility prior to removing samples from biocontainment. Lung supernatants were used in performance of a multiplex ELISA (Bio-rad Bio-plex Pro™ human cytokine 27-plex kit) according to the manufacturer’s instructions to assess changes in expression of human cytokines: IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, IP-10, FGF basic, Eotaxin, G-CSF, GM-CSF, IFN-γ, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF. Values for cytokines where cross reactivity between human detection reagents and mouse cytokines could confound data (e.g. VEGF, IL-13) were excluded from the analysis. Levels of each cytokine were quantified by generating a standard curve using the standards provided in the kit. Values were generated by linear regression to the standard curve as recommended by the manufacturer and as described62 (link). Values that were below the extrapolated range (
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