Multiplexing GPCR Signaling Pathways with Spectrally Resolved Fluorescent Dyes

Simultaneously measure calcium signaling and GIRK activation using Fluo-Gold and Thallos on the CLARIOstar® Plus.

Understanding the complex interactions of G protein-coupled receptor (GPCR) signaling pathways is critical for advancing drug discovery and biomedical research.

In this collaborative application note, ION Biosciences and BMG LABTECH demonstrate the successful detection of two distinct GPCR signaling casecades using spectrally resolved fluorescent dyes. By combining Fluo-Gold AM and Thallos AM on the CLARIOstar® Plus platform, researchers can monitor calcium release induced by Gq agonism and potassium channel activitation by the beta/gamma subunits of Gi GPCRs in real time within the same well.

A multiplexed approach can help you uncover biased ligands and simultaneously promotes faster outcomes in your GPCR discovery campaign.

KEY BENEFITS

Monitor Multiple Signaling Pathways Simultaneously

Measure two distinct GPCR signaling pathways in the same well for more complete SAR.

Spectral Resolution

The PHERAstar FSX provides ultimate flexibility for discriminating between dyes.

Double the Data Output

Capturing two data points simultaneously saves time and resources.

Reduce Profiling Time

Generate richer datasets in fewer experiments to accelerate compound characterization and screening workflows.

Assay Principle

Cells expressing M3 and D2 receptors along with GIRK channels are loaded with Fluo-Gold AM and Thallos AM dyes.

Stimulation with acetylcholine activates the M3 receptor and triggers calcium release, while dopamine activates the D2 receptor and induces potassium flux through GIRK channels. Using spectrally resolved fluorescent dyes, both signaling pathways can be measured simultaneously in real time.

This approach can be translated to other GPCR secondary messenger and fluorescent sensor pairs within our portfolio.