Concentrations of plasma cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α) were determined in all mice (n = 163, excluding n = 4 used in flow cytometry studies) as previously described (9 (link)). Concentrations of cytokines and chemokines (identical to those measured in plasma, with the addition of IL-33) in 2 cm of ileum tissue were determined in the 153 mice not used for transmission studies (n = 10) or flow cytometry studies (n = 4). Ileum tissue was collected at necropsy, weighed, snap-frozen, and stored at −80° C until processing for protein isolation using the Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA), as described (19 (link)). Briefly, tissue samples were washed with lysis solution and lysed by probe sonication (Branson SFX150, Branson, Danbury, CT) followed by water bath sonication for 3 min and centrifugation at 4500 × g for 4 min at 4°C. After measuring protein concentration using a Pierce bicinchoninic acid assay (Thermo Fisher Scientific), cytokine and chemokine profiles were determined for each ileum sample (in duplicate) by a Bio-Plex Pro Luminex assay.
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