Cellomics HCS Reagent Kit for detection of cyclooxygenase-2 (COX-2) enzyme.

control	treat cells	detect COX-2	analyze	conclude

Description

The Cellomics COX-2 Activation Kits are for detecting and measuring cytoplasmic induction of the COX-2 enzyme. 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 (IFNg), 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 IFNg was quantitatively assayed using the Cellomics COX-2 HCS Reagent Kits, the Cellomics ArrayScan HCS Reader (Figure 1) and the Compartmental Analysis BioApplication Software Module. Cells labeled using this kit also can be imaged by fluorescence or confocal microscopy.

Non-treated cells	Treated cells	Dose response curve
Figure 1. COX-2 induction after treatment with LPS and interferon gamma (IFNg). 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 (center); dose response curves (right) of COX-2 expression to IFNg. EC50 = 174+/-0.03 ng/ml LPS; 100+/-0.03 ng/ml IFNg.

Related Poster Presentation

	Kay Opperman, Eric Hommema, Brian Webb, and Richik N. Ghosh (2008) Combining Multiplexed, High-Content Analysis with Antibody Arrays to Monitor and Correlate Intracellular and Extracellular Inflammatory Responses.
	Kay Opperman, and Richik N. Ghosh (2007) New High Content Screening and Cell-Based Assays for Inflammation.

Kit Contents

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

Choose "Order Online" in table to see pricing and order online (U.S. only).

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