Bioplex multiplex

The distal section of colon was excised and cut into 1 cm² sections. Tissues were washed in PBS containing penicillin and streptomycin, and the weight of each section was recorded. The colon section was placed in complete RPMI medium 1640 (10% FBS, 1% penicillin and streptomycin) and cultured at 37 °C for 24 h. The supernatants were harvested and cytokines were measured by ELISA from BioPlex Multiplex (Bio-Rad) according to the manufacturer's instructions. CXCL-5 was measured using an ELISA Kit (abcam).

Serum samples were screened to quantify human cytokines, chemokines, and growth factors with pre-formed kits by Bioplex multiplex (Bio-Rad, Hercules, CA, USA, cat # M500KCAF0Y). Samples were diluted (1:4) and 50µl were used, according to the manufacturer’s instructions. The concentration of IL-1RA, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, basic fibroblast growth factor (FGF), eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), interferon-γ inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1) α, MIP-1β, C–C motif chemokine ligand 5 (CCL5)/RANTES, TNF-α, platelet-derived growth factor (PDGF-BB) and vascular endothelial growth factor (VEGF) were determined according to the manufacturer’s protocol as previously described (30 (link)). The magnetic bead-based assay was performed on a Bio-Plex 200 System (Bio-Rad, Hercules, CA, USA).

BALB/cJ mice (5 mice per group) in the immunogenicity study were euthanized three weeks after the final immunization and spleens from each mouse were harvested and processed into single-cell suspensions. Single-cell suspensions were prepared from whole spleens by cutting each spleen into small pieces followed by pressing through a 70 µM filter using the rubber end of a sterile 3 ml syringe plunger. Cells were pelleted by centrifugation and red blood cells were lysed using RBL Lysis Buffer (Cat# R7757 Sigma; St. Louis, MO) according to the manufacturer’s recommendation. Splenocytes were washed twice in RPMI-1640 containing 5% FBS (Cat#25-514 Genesee; San Diego, CA) and 1% penicillin/streptomycin (Cat#15140122 ThermoFisher; Waltham, MA) before counting and plating. Splenocytes were plated in 48-well plates (2.5 x 10⁶ cells/well) and cultured in media alone (39 ) or media containing EDIII (25 μg/ml) in a total volume of 500 µl for 72 hours at 37°C and 5% CO₂. Supernatants were collected and measured for IL-4, IL-5, IL-17, and IFN-γ using the Bioplex Multiplex (Biorad; Hercules, CA) according to the manufacturer’s instructions. EDIII-induced cytokine responses were determined by subtracting the cytokine value from cells cultured in media from the cytokine values measured in cells stimulated with EDIII.