Bioplex manager version 4

Cell supernatant collected following treatment with single or multiple TLR ligands was tested for inflammatory mediator accumulation. The quantification was performed by in house multiplex bead immunoassay as previously described [14 (link)]. Culture supernatants were simultaneously assessed for the presence of the following molecules: IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-12, IL-16, GM-CSF, IFN-β, IFN-γ, CXCL10 (IP-10), CCL7 (MCP-2), CXCL9 (MIG), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL12 (SDF-1), TGF-β and TNF. In this assay microbeads (R&D Systems) dyed with different concentrations of fluorophores were used in order to create distinct sets. Each set was then coated with an antibody specific for one of the analytes (R&D Systems) and the captured analyte was detected with a biotinylated antibody followed by incubation with Streptavidin-phycoerithrin (S-PE). Plates were read using the Luminex 100 system (Luminex Corp., USA) and data analysed by Bioplex Manager Version 4.0 software (BioRad, UK). Lower detection limits for this assay were 5.3 pg/ml (IFN-β, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-16, CXCL10, CCL8, CXCL9, CCL3, CCL4, CCL5, CXCL12, TNF), 2.6 pg/ml (IL-12 and TGF-β), 5.6 pg/ml (IL-6) and 2.8 pg/ml (GM-CSF).