Luminex magpix multiplex reader

Whole blood samples were aliquoted and mixed 1:1 with HBSS with bovine albumin [final concentration 0.2 mg/ml] alone or HBSS with albumin and LPS from Escherichia coli 026:B6 [final concentration 100 ng/ml] (Sigma-Aldrich, St. Louis, MO, USA). Aliquots were then incubated on a rocker for two hours at 37° C and 5% CO₂. Following incubation, whole blood was centrifuged at 1800 × g for 10 minutes at 4° C, and cell-free supernatant was collected and stored at −80° C until batch processing to reduce the degree of intra-assay variability. The concentrations of interleukin (IL)-1β, IL-6, IL-8 (CXCL8), IL-10, IL-12 p70, interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), macrophage inflammatory protein-1α (MIP-1α, CCL3), monocyte chemotactic protein-1 (MCP-1, CCL2), and interferon gamma-induced protein-10 (IP-10, CXCL10) were determined using Milliplex® Multiplex kits (EMD Millipore, Billerica, MA, USA) on the Luminex^® MAGpix Multiplex reader. Cytokine assay sensitivities (minimum detectable concentrations, pg/mL) are provided in Supplemental Table 1.