Luminex kit

Blood samples were centrifuged for 10 min at 600 ×g, and serum was immediately aliquoted and stored at 80°C. The following 11 cytokines were measured with a Luminex kit according to the manufacturer's instructions: IL-1β, IL-2, IL-6, IL-8, IL-10, IFN-γ, MCP-1, MIP-1β, TNF-α (Merck Millipore, Billerica, Massachusetts, USA, PRCYTOMAG-40K-09C), TGF-β (Merck Millipore, Billerica, Massachusetts, USA, PRCYTOMAG-40K-09C), and IP-10 (EPX01A-40284-901, Carlsbad, CA, USA). After thawing the samples on ice and su cient mixing, 25 μl of supernatant was loaded into each well of a 96-well plate and mixed with 25 μl of assay buffer and 25 μl of magnetic beads. The plates were incubated with agitation overnight at 4°C. After washing, 25 μl of detection antibody was added to each well and the plate was incubated 1 h at room temperature (RT). Then, 25 μl of streptavidin-PE was added to each well and incubated for 30 min at RT. Next, 150 μl sheath uid was added to each well after washing. Plates were read on a Luminex ® 200 (Bio-Rad, CA, USA) and the data analyzed for median uorescent intensity (MFI) using a ve-parameter logistic method for calculating analyte concentration.