Bio plex pro mouse cytokine 23 plex immunoassay

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).

The levels of inflammatory mediators in BAL fluid were assessed by the Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay (Cat# M60009RDPD, BioRad, Hercules, CA, USA) via Luminex. Experiments were completed following the manufacturer’s instructions, and the results were presented as pg/mL in the samples.

For multiplex serum cytokine detection, mouse blood samples were obtained via retro-orbital blood collection and were then centrifuged at 12,000×g for 15 min at 4 °C. The supernatants were collected, and the concentrations of 23 mouse cytokines were determined using a Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay (Bio-Rad, Hercules, Cat. M60009RDPD/64465244) according to the manufacturer’s specifications.

The levels of interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α in the peritoneal fluids, collected from the control mice and the LPS-stimulated mice, which were post-challenged with and without KPC160111 or KPC160132, were quantified using the Bio-Plex Pro Mouse Cytokine 23-Plex Immunoassay (Bio-Rad; Model: M60009RDPD) according to the instruction manual.