Procartaplex mouse cytokine and chemokine panel

Cytokine analysis of plasma and ileal homogenate was performed for interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-12 p70, interferon (IFN)-γ, CXCL1 (C–X–C chemokine ligand 1), and IL-10 with a Luminex-based ProcartaPlex Mouse Cytokine and Chemokine Panel (eBioscience, San Diego, CA, USA), per the manufacturer’s instructions. Small intestinal samples were homogenized using a bead beater (Next Advance INC, Troy, NY, USA) in a buffer containing phosphatase and protease inhibitors (Millipore, Burlington, MA, USA) and phenylmethanesulfonylfluoride (Sigma-Aldrich). Samples were run in duplicate on a BioPlex 200 (Bio-Rad, Hercules, CA, USA), and results were calculated on the basis of a seven-point, five-parameter logistic standard curve for each analyte. Final cytokine levels were normalized to total protein concentration (mg/mL) and logarithmically transformed using log(x) (or log(x + 1) when values were <1) to reduce skew in data.

Spleen cells were cultured with the mitogen concanavalin A (Con A) (Sigma-Aldrich, St. Louis, MO, USA) in microtitrate plates at 37 °C for 48 h. The supernatants were stored at −70 °C. The ProcartaPlex^® Mouse cytokine and chemokine panel (eBioscience, San Diego, CA, USA) was used to measure the levels of Eotaxin/CCL11, granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine growth-regulated protein alpha (GRO-α/KC/CXCL1), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17A, IL-18, IL-22, IL-23, and IL-27, interferon gamma-inducible protein 10 (IP-10/CXCL10), monocyte chemotactic protein-1 (MCP-1/CCL-2), MCP-3/CCL7, macrophage inflammatory protein-1α (MIP-1α/CCL3), MIP-1β/CCL4, MIP-2/CCL8, RANTES/CCL5, and tumor necrosis factor-α (TNF-α) according to the manufacturer’s instructions by using the Luminex xMAP instrument (Luminex-Corporate, Austin, TX, USA). Values extrapolated beyond the calibration curve (cc) and values outside the cc range (below, above) were discarded. Levels of GRO-α/KC/CXCL1, IL-1β, IL-5, IL-9, IL-12p70, IL-22, IL-23, IL-27 were below the limit of detection and are therefore not presented.