Cytokine specific multiplex beads

Tissue homogenates were gamma-irradiated (5 megarads) prior to analysis at BSL2. Samples were processed according to manufacturer instructions and then analyzed using a Bio-Plex 200 system (Bio-Rad, Hercules, CA). Briefly, the tissue homogenates were centrifuged for 10 minutes at 1,000 rpm at 4°C to remove cellular debris. The cleared supernatants were aliquoted into 96-well plates in pre-determined wells; this plate was centrifuged at 1,250 rpm to remove any remaining debris. The supernatant was transferred to a 96-well flat bottom plate and processed for use on the Bio-Plex system. The cytokines were coupled to cytokine-specific multiplex beads (Bio-Rad) following the manufacturer's instructions using the pre-designed assays Bio-Plex Pro mouse cytokine 23-plex immunoassay. This panel measures the concentrations of cytokines and chemokines and includes interleukin (IL)-1α/β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40, p70), IL-13, IL-17, eotaxin (CCL11), IFN-γ, KC (CXCL1), monocyte chemoattractant protein (MCP)-1/(CCL2), granulocyte colony-stimulating factor (G-CSF/CSF3), granulocyte macrophage colony-stimulating factor (GM-CSF/CSF2), macrophage inflammatory protein (MIP)1α/β (CCL3/CCL4), tumor necrosis factor (TNF)-α and RANTES (CCL5).

Cell culture supernatants and mouse sera were processed according to the manufacturer’s instructions and then analyzed using a Bio-plex 200 system (Bio-Rad, Hercules, CA). Briefly, the samples were filter sterilized and subsequently centrifuged for 10 minutes at 450 × g at 4°C to remove debris. The resulting supernatants were collected and aliquoted into 96-well plates and processed for analysis on the Bio-plex system. The cytokines were coupled to cytokine-specific multi-plex beads (Bio-Rad) in the Bio-Plex mouse cytokine immunoassay following the manufacturer’s instructions. The pre-designed assay kit measured the concentrations of cytokines/chemokines including interleukin (IL)-1α, IL-6, IL-10, IFN-γ, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor (TNF)-α.