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Automated Fluorescence Imaging of GPCR Dependent Second-Messenger SystemsDownload
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January 21, 2014
Authors: Paul Held and Peter Banks, BioTek Instruments, Inc., Winooski, Vermont; Anne Marie Quinn and Thom Hughes, Montana Molecular, Bozeman, Montana
Cell signaling involves an integrated, multi-faceted series of second-messenger systems that act in concert to transfer cell surface receptor mediated signals to a wide variety of intracellular processes. G-Protein Coupled Receptors (GPCRs) are the most notable initiators of cell signaling cascades and are the targets of a large amount of the drug discovery effort. Drug discovery screening of compound libraries relies on the ability to rapidly make assay determinations on large numbers of samples. At the same time, phenotypic information is also desired to assess the true cellular response. Towards that end, having multiple sensors capable of simultaneously detecting different signaling components of the GPCR pathways are of particular importance.
Here we describe the use of a combination microplate reader and imager to detect changes in second messenger levels using a series of genetically encoded fluorescent sensors. The multimode microplate reader is capable of digital microscopy and conventional microplate detection. New genetically-encoded biosensors for diacylglycerol (DAG), PIP2 phospholipid, Ca++, and cyclic adenosine monophosphate (cAMP) change fluorescence in response to changes in analyte concentration, and can be multiplexed in a single live cell assay [1,2,3]. This combination of reader/imager and second messenger biosensor has the capability of making both rapid analytical determinations, as well as phenotypic assessments in live cells.
Coordinated GPCR receptor and phospholipase C (PLC) activation were assessed using green and red fluorescent sensors for DAG, PIP2, cAMP and Ca++ in a multiplexed format. A number of sensors capable of either a greater “upward” or a lesser “downward” amount of fl uorescence in the presence of their specific analyte were tested. For example, the addition of carbachol to HEK293 cells transfected with “Upward DAG” green plasmid resulted in a rapid increase in green fluorescence, with fluorescence levels returning to baseline within 15 seconds from the addition of stimuli, while the response of the “DownwardDAG” green sensor is a loss of fluorescence that is regained over time. Object cell counting of green cell fluorescence demonstrates a 4-fold increase in the green positive cell number with an upward calcium specific probe. Counting analysis of green and red fluorescence multiplexed reactions tracking DAG against a red control results in a 15% increase in the Green/Red ratio, while the use of a Downward Green DAG probe in conjunction with an Upward Red Ca++ probe resulted in a 50% decrease in the Green/Red ratio with carbachol stimulation. Image and analytical analysis of results will be presented.