Meso scale discovery

Total cell lysates from ileal samples were prepared in ice-cold lysis buffer (500 mM NaCl, 50 mM Tris pH 7.5, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS, 1 mM protease inhibitor). Protein concentrations were determined by bicinchoninic acid assay (Sigma-Aldrich, Overijse, Belgium). Cell extracts were subjected to cytokine analyses using the MSD mouse pro-inflammatory V-plex assay containing antibodies for IL1β, IL10, IFNγ and TNFα. These assays were performed according to manufacturer’s instructions using the MSD Quickplex SQ 120 instrument and MSD Discovery Workbench data analysis software v4.0 (Meso Scale Discovery^TM; MSD, Rockville, MD, USA).

Serum samples from clinic visits, thawed on ice, were analyzed using a customized Meso Scale Discovery^TM immunoassay, MSD Human Custom Cytokine 9-Plex US. This technology includes biotinylated and conjugated SULFO-TAG detection antibodies to measure nine different analytes in one microtiter well. Pristine serum from each subject at each time point (50ul) was thawed and analyzed in duplicate for nine cytokines per manufacturer recommendations: interferon (IFN)-α2a, interleukin (IL)-6, IL-17, IL-1β, interferon gamma-induced protein (IP)-10, monocyte chemotactic protein (MCP)-1, IFN-γ, IL-1α, and IL-1ra (IL-1 receptor antagonist).