Colon Tissue Biopsy and Cytokine Analysis

Collection of colon tissues and processing was performed as previously described (Henderson et al., 2015 (link)). Briefly, a 1 cm² colon tissue biopsy was collected from ASF and CONV mice (n=4 per group), placed in PBS to remove intestinal contents and transferred to a tube containing RPMI plus gentamicin (50 μg ml⁻¹), penicillin (200 IU ml⁻¹) and streptomycin (200 μg ml⁻¹) at room temperature with shaking at 280 rpm. After 30 min, tissues were transferred to 96-well plates containing RPMI plus gentamicin (50 μg ml⁻¹), penicillin (200 IU ml⁻¹), streptomycin (200 μg ml⁻¹), L-glutamine (2 mM), 2-mercaptoethanol (0.05 mM) and pyruvate (1 mM), and incubated at 37°C in 5% CO₂. After 24 h, supernatants were harvested and frozen at −20°C until further use. Levels of proinflammatory markers IL-1β, IL-12, IFNγ and TNFα were assessed using a multiplex bead assay and Bio-Plex 200 multiplex reader (Bio-Rad, Hercules, CA).