Characterizing GPCR Activation Using Automated Live Cell Imaging
Poster Jul 24, 2017
Joe Clayton and Peter Banks
G protein coupled receptor (GPCR)-mediated pathways are critical for cells to respond to intercellular and environmental cues, and are a major focus of drug discovery efforts, particularly for cancer treatment. The molecules that activate GPCRs, and the resulting signaling cascades triggered by associated G proteins, are diverse. Fluorescent dyes and biosensors can be used to monitor changes in second messenger levels, including Ca2+ and cyclic AMP (cAMP), in response to GPCR activation. Here we describe a live cell imaging based approach to detect GPCR activation using the Lionheart™ FX Automated Live Cell Imager and Gen5™ Microplate Reader and Imager Software. This method provides a large assay window and improved sensitivity over methods relying on total fluorescence intensity measurements. Dual in-line dispense tips enable addition of GPCR agonists with continuous monitoring of cellular response. Additionally, an image capture rate of up 20 frames per second enables characterization of rapid GPCR kinetics.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.