ELISA for TNF-α, IL-6, and MDA

Jejunal mucosa was weighed to 0.5 g and homogenized (Tissuemiser, Thermo Fisher Scientific Inc., Rockford, IL) on ice in 2 mL PBS. Sample preparation for analysis was followed as previously described by Chen et al. (2017). Homogenized samples were centrifuged at 15,000 × g for 30 min and then the supernatant was collected. The protein amount in supernatant was measured using Pierce BCA Protein Assay Kit (23225#, Thermo Fisher Scientific Inc., Rockford, IL) to determine concentrations of TNF-α, IL-6, and MDA per mg of protein in the jejunal mucosa.

Concentration of TNF-α, IL-6, and MDA in plasma and jejunal mucosa were measured using ELISA kits for TNF-α and IL-6 (R&D Systems, Minneapolis, MN) and Thiobarbituric Acid Reactive Substance (TBARS) Assay Kit for MDA (Cell Biolabs, Inc., San Diego, CA) following the manufacturer’s protocols. These kit analysis was followed as previously described by Chen et al. (2017).