COX-2 Activation Kit

These kits contain a monoclonal mouse anti-COX-2 antibody, a goat anti-mouse secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence detection of COX-2 for high-content screening (HCS) assays. This assay is primarily for immune cells, specifically macrophages, which are key mediators of the innate immune response and cytokine production.

Once stimulated with lipopolysaccharide (LPS) and interferon gamma (IFNγ), or other harmful stimuli, immune cells defensively produce cytokines, prostaglandins, chemokines, and reactive amines. Prostaglandins (PGs), produced by the cyclooxygenase (COX) enzymes are critical in the immune response to induce vasodilation, vasoconstriction, pain, and fever. Prostaglandins are produced by converting arachidonic acid to prostaglandin G2 (cyclooxygenase) and prostaglandin G2 to prostaglandin H2 (peroxidase), which is then further converted to other prostaglandins by cell-specific enzymes. Of the three forms of COX, COX-2 is inducible upon stimulus, and its product PGE₂ is important in signaling and inflammation.

COX-2 in cells induced with LPS and IFNγ was quantitatively assayed using the Cellomics COX-2 HCS Reagent Kits, the Cellomics ArrayScan® HCS Reader (Figure 2) and the Compartmental Analysis BioApplication Software Module. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

 
Figure 2. COX-2 induction after treatment with LPS and interferon gamma (IFNγ). RAW 264.7 murine macrophages were challenged with 500 ng/ml LPS and 100 ng/ml mouse interferon gamma overnight (20-24 hours) to measure cytoplasmic induction of COX-2. Cells were then fixed and stained with primary antibody towards COX-2 and DyLight 549 Goat anti-Mouse secondary antibody (orange). Cells were also labeled with Hoechst Dye for nuclear staining (blue). Untreated cells (left); treated cells (right); dose response curves (bottom) of COX-2 expression to IFNg. EC₅₀ = 174 ± 0.03 ng/ml LPS; 100 ± 0.03 ng/ml IFNγ.