Assay principle

The kit is based on a competitive format involving a specific antibody labelled with Cryptate (donor) and cGMP coupled to d2 (acceptor). cGMP produced by cells competes with d2-labelled cGMP for binding to monoclonal anti-cGMP Eu Cryptate.

Assay protocol

The cGMP assay features a streamlined protocol with only two incubation steps:

- Cell stimulation by target compounds

- cGMP detection using HTRF reagents.

This protocol requires only a single, one-hour incubation period following cell stimulation.

Figure 1: 300 to 20,000 RFL-6 rat lung fibroblasts were plated per well in a low volume 384-well microplate. Cells were incubated either in the presence or absence of SNAP and cGMP, quantified using the HTRF cGMP kit following the standard assay protocol.

Figure 2: 10,000 RFL-6 rat lung fibroblasts were plated per well in a low volume 384-well microplate and stimulated with increasing concentrations of ANP. cGMP levels were calculated using the HTRF cGMP kit, following the standard assay protocol.

Increases in IW-1973 increases both cGMP production and VASP phosphorylation. IW-1973 is a sGC stimulator. cGMP production and VASP phosphorylation were measured using the HTRF assays.

Adapted from Tobin J.V, et al (2018). Pharmacological characterization of IW-1973, a novel soluble guanylate cyclase stimulator with extensive tissue distribution, anti-hypertensive, anti- inflammatory, and anti-fibrotic effects in preclinical models of disease. J Pharmacol Exp Ther 365(3):664-675

Nitric Oxide signaling pathway

Nitric Oxide is a key effector protein that - among other roles - is involved in cardiovascular hemostasis and vascular relaxation. In the event of a vascular stress, NO is synthesized at its location. It binds to sGC on the environing tissues, triggering the downstream decrease of Ca2+ and increase of cGMP, which in turn alters the activity of a collection of protein kinase G, cyclic nucleotide-gated ion channels and phosphodiesterases, leading to the vasodilatation of nearby vessels, blood flow increase and lowered tension.