Bioplex assay

Sera from each mouse were collected for measurement of cytokines using Bio-plex Pro Mouse Th17 assay (Bio-rad, CA, USA) which included the following cytokines: IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-31, IL-33 and MIP-3α according to the manufacturer’s instructions. The Multiplex bead working solution was diluted from 25× stock solution beads and 50 μl of it was added into each well followed by 50 μl of sample. Each cytokine standards and samples were assayed in duplicate as provided by manufacturer. Samples with microbeads were incubated at room temperature on a magnetic microplate shaker for 30 min. After incubation, Bio-Plex detection antibody working solution was then added, washed 3× with Bio-Plex wash buffer and finally 1× streptavidin-PE was added before reading the plate on the Bio-Plex 200 system (Bio-Rad). Cytokine concentrations from each tissue homogenates were calculated based on each cytokine standard curve.