Milliplex map mouse cytokine chemokine magnetic bead panel immunology multiplex assay

The concentrations of various cytokines, chemokines, and other factors in serum and synovial fluid were measured by MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel-Immunology Multiplex Assay from Millipore (Billerica, MA, USA) according to the manufacturer’s instructions. Serum and synovial fluid were incubated with 100 μg of vancomycin for 30 min; the TV of the solution was then volume-adjusted with assay buffer. A 96-well filter plate was loaded with either 50 μl of a prepared standard solution or 50 μl of serum and synovial fluid. The solutions in the plate were then incubated with capture antibody–conjugated beads at ±800 rpm for 30 min, at room temperature, and in the dark. Wells were vacuum-aspirated and washed with 100 μl of wash buffer three times. Samples were incubated with 25 μl of biotinylated detection antibody at ±800 rpm for 30 min, at room temperature, and in the dark. After three more washes, 50 μl of streptavidin-phycoerythrin was added to each well and incubated for 10 min at ±800 rpm at room temperature and in the dark. After one final wash, the beads were resuspended in 125 μl of sheath buffer for measurement with the Luminex 200 (Luminex, Austin, TX, USA). PCA was performed with QStudioMetrics (https://github.com/gmrandazzo/QStudioMetrics).