Multiplex Cytokine Measurement in Serum

Measurement of cytokines and chemokines in serum samples was carried out as previously reported^{18 (link)}. The serum levels of IL-6, IP-10, MCP-1, and MIP-1β were determined using a MAGPIX Milliplex kit (Merck, Darmstadt, Germany) according to the manufacturer’s instructions. Briefly, 25 μL serum samples, standards, and controls were added to a 96-well plate comprising an equal amount of assay buffer for serum samples or serum matrix for standards and controls. Next, magnetic beads coated with antibodies against the target cytokines were added to each well, and the plates were incubated on a plate shaker overnight at 4 °C. After washing with washing buffer in the kit, the samples were reacted with biotinylated detection antibodies for 1 h and then with streptavidin–phycoerythrin for 30 min. After washing and the addition of loading buffer from the kit, the samples were analyzed by the MAGPIX system (Luminex, Austin, TX, USA).