Description
Prostaglandin E2 (PGE2) is produced in multiple cell types from PGH2, a primary product of the arachidonic acid metabolism, via the prostaglandin synthase. PGE2 displays several biological functions, such as vasodilatation and smooth muscle relaxation, and is involved in pro- and anti-inflammation pathways. PGE2 production is a commonly used method for the detection of COX-1 and COX-2 modulation. The PGE2 kit is a highly sensitive method for quantifying PGE2, either in cell supernatant or directly in whole cells.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The PGE2 assay is based on the competition principle, where native PG2 produced by cells and d2-labelled PGE2 compete for binding to a monoclonal anti-PGE2 antibody labelled with Europium Cryptate. The assay can be run with several types of samples, such as cell supernatants or purified enzymes.
Assay Protocol
The PGE2 assay features a streamlined protocol with only 1 incubation step after sample and PGE2 detection reagents dispensing. This protocol requires a single 5-hour incubation period at RT.
PGE2 modulation upon Indomethacin inhibitor treatment
One benefit of the HTRF PGE2 assay is its ability to quantify PGE2 in the presence of cells without sacrificing assay performance. The results comparing direct cell-based versus supernatant transfer did not differ significantly. Monocytes were stimulated to produce PGE2 in the presence or absence of indomethacin, a known inhibitor of PGE2 production. The IC50 of indomethacin determined using HTRF technology (1.0 ± 0.4 nM) was in agreement with previously published data.
