A multiplex bead array assay was used to examine plasma cytokines levels. The detailed procedures for detection of soluble cytokines using the multiplex bead array assays have been previously reported (Ho et al., 2015, 2017). We examined 29 cytokines using a customized human cytokine magnetic bead panel (Bio-Rad; Yu-Shing Biotech., Ltd, Taipei, Taiwan) according to the manufacturer’s instructions (Bio-Rad; Genmall Biotechnology Co., LTD., Taipei, Taiwan). The examined cytokines were IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-17A, IL-23, IL-25, IL-31, interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), soluble CD40 ligand (sCD40L), IFNγ-induced protein 10 (IP10), monocyte chemoattractant protein 1 (MCP1), macrophage inflammatory protein 1-alpha (MIP1α), MIP1β, regulated upon activation, normal T cell expressed and secreted (RANTES), eotaxin, fibroblast growth factor (FGF), granulocyte colony-stimulating factor (GCSF), Granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor-BB (PDGF-BB), and vascular endothelial growth factor (VEGF). The median fluorescence intensities were assessed using a Bio-Plex 200 instrument (Bio-Rad) with Bio-Plex Manager software version 6.0 (Bio-Rad). Study samples were assessed in duplicates and the duplicate measurements were averaged for statistical analysis. Standard curves were created from duplicate values and all samples were analyzed as single determinants. All analyses were performed in one batch using kits from the same production lot.

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