title: Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging.
abstract: Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of model cell lines and primary dorsal root ganglia neurons.
authors.affiliation: Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia, michelle.halls@monash.edu.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Halls
authors.forename: Daniel P
authors.initials: DP
authors.lastname: Poole
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Ellisdon
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Nowell
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D018929:Cell Culture Techniques; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005727:Ganglia, Spinal / Q000166:cytology; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000378:metabolism*; D061986:MCF-7 Cells; D008297:Male; D057054:Molecular Imaging / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection