Bio plex 200

Cytokines in the Human Th17 Cytokine Panel (listed in Table 2) were measured using Luminex xMAP technology on the Bio‐Plex 200 platform and analyzed with Bio‐Plex Manager 5.0 software (Bio‐Rad Laboratories Ltd, Hercules, CA). Serially diluted standards and test sera diluted 1 in 4 in sample diluent were added in 50 μL to a plate containing biotin‐labeled antibody‐coupled magnetic beads for each of the 15 cytokines in the panel. Samples were incubated at room temperature on a plate shaker at 300 rpm for 30 min. Following washing, secondary detection antibodies were added in 25 μL to each well and incubated as described above. After a further wash, streptavidin‐phycoerythrin (streptavidin‐PE) was added in 50 μL, and the plate incubated at room temperature on an orbital shaker at 300 rpm for 10 min. Assay buffer (125 μL) was added to each well before analysis on the Bio‐Plex 200 suspension array system. Fluorescent intensities obtained for the test samples were converted to pg/mL using the standard curves for each cytokine. Out‐of‐range cytokine concentrations were assigned a value corresponding to the minimum or maximum detectable concentration of the standard. The lower limit of detection varied for each cytokine (Table 2). According to the manufacturer's specifications, the intra‐ and inter‐assay coefficients of variation were <10 and <20%, respectively, for all cytokines.