Luminex is100 system

PBMCs from 5 different blood donors (Precision Bioservices) were cultured in the presence of 30 or 100 μg/mL AvFc for 3 days, and the concentrations of interleukin (IL)-1α, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF or CSF2), granulocyte colony-stimulating factor (G-CSF or CSF3), interferon gamma (IFN-γ), IFN-α2, interferon-γ-inducible protein-10 (IP-10), monocyte chemoattractant protein-1(MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, tumor necrosis factor (TNF)-α, and TNF-b were evaluated in culture supernatants by the Luminex IS100 system (Millipore) using the MILLIPLEX Human Cytokine/Chemokine 26-Plex Kit according to the manufacturer’s instructions. Data were generated with the xPONENT software. Individual ELISAs were used to evaluate the amounts of IL-1β, IL-6, and IL-8 in cell-culture supernatants collected from PBMC cultures after 24 h of incubation in the presence of AvFc (30 or 100 μg/mL) or controls (10 μg/mL ConA or PBS). ELISA Ready-SET-Go! Kits were purchased from eBioscience and used in these experiments following the manufacturer’s instructions. The experiments were repeated for a total of at least two times.