Multiplexing the production of two G protein-coupled receptor second messengers using spectrally resolved fluorescent dyes

Introduction

With about 1000 members, G protein-coupled receptors (GPCRs) represent the largest superfamily of proteins that serve as receptors localized to cell surface membranes. They share conserved elements and employ similar hetero-trimeric proteins to initiate production of second messengers to participate in a wide variety of physiological responses. Due to their physiological importance, GPCRs are the target of about 1/3 of approved drugs¹.

Despite all we have learned about GPCR’s and their signalling there is still much to learn, for example, the complex interactions between receptors and the signalling cascades they initiate¹. To further our understanding of these important responses ION Biosciences has produced several tools to detect GPCR stimulated second messengers. They have used a variety of fluorophores that span the visible spectrum as a part of these tools thus enabling the detection of multiple signalling events simultaneously.

Here we show how the detection of calcium and thallium flux can be detected simultaneously. Using the CLARIOstar Plus with LVF monochromator allowed easy adjustment of the detection wavelengths to optimize the multiplex detection.

Assay principle

Results & Discussion

Initial studies showed that all dyes tested from ION Biosciences worked very well individually (data not shown), but we wanted to test multiplexing capacity. Figure 2 shows that the default settings available on the CLARIOstar Plus for the relevant fluorophores for Thallos AM and Fluo-Gold AM work well but exhibit some bleed-through (Fig. 2A). A small adjustment of the detection wavelengths, easily achieved because of the LVF monochromator, decreases the bleed-through (Fig. 2B).