Category: Assay Development and Screening

1128-D - Automated imaging-based techniques for detailed characterization of GPCR signaling pathways

Tuesday, February 6, 2018
5:00 PM - 6:00 PM

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. The molecules that activate GPCRs, and the resulting signaling cascades triggered by associated G proteins, are diverse. Activation of the Gq sub-family of G proteins increases phospholipase C (PLC) activity, converting phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), both of which function as second messengers. IP3 triggers release of intracellular Ca2+ stores, which regulate calcium-dependent proteins, while membrane-bound DAG activates the signal cascade via protein kinase C. 

Here we describe an imaging-based approach using the Lionheart FX automated imager and R-GECO Ca2+ and DAG fluorescent biosensors from Montana Molecular to characterize the IP3/DAG signaling pathway. Dual in-line reagent injectors and an image capture rate of up to 20 frames per second supports uninterrupted monitoring of rapid cellular responses with high temporal resolution. The ability to multiplex biosensors provides detailed kinetic profiles of Ca2+ flux and DAG levels within the same population of cells.

Gen5 background subtraction and image analysis tools enable detection of changes in Ca2+ and DAG levels that is up to 8 times more sensitive than techniques relying on bulk fluorescence measurements.  Additionally, this imaging-based method enables detailed characterization of cellular responses following GPCR activation, including single-cell kinetic profiles, dynamics of Ca2+ cycling, and subcellular localization of second messengers.

Xavier Amouretti

Manager, Product Marketing
BioTek Instruments
Winooski, VT

Xavier Amouretti is the Manager of Product Marketing at BioTek Instruments, Inc. Before joining BioTek USA, Amouretti was working in Europe in the field of ELISA automation and microplate-based luminescence detection. His background is in Biochemistry and Neuroscience.