Real-Time Imaging of Nitric Oxide Signals in Individual Cells Using geNOps
Nitric oxide (NO•) is a versatile signaling molecule which regulates fundamental cellular processes in all domains of life. However, due to the radical nature of NO• it has a very short half-life that makes it challenging to trace its formation, diffusion, and degradation on the level of individual cells. Very recently, we expanded the family of genetically encoded sensors by introducing a novel class of single fluorescent protein-based NO• probes—the geNOps. Once expressed in cells of interest, geNOps selectively respond to NO• by fluorescence quench, which enables real-time monitoring of cellular NO• signals. Here, we describe detailed methods suitable for imaging of NO• signals in mammalian cells. This novel approach may facilitate a broad range of studies to (re)investigate the complex NO• biochemistry in living cells.
Key wordsFluorescence microscopy Genetically encoded probes Nitric oxide imaging Single cell analysis
The authors thank the scientific advisory board of NGFI (Next Generation Fluorescence Imaging GmbH, Graz, Austria, http://www.ngfi.eu/) for their support.
Sources of Funding
This work is supported by Nikon Austria within the Nikon-Center of Excellence Graz. The researchers are also supported by the Ph.D. program Metabolic and Cardiovascular Disease (DK-W1226) of the Medical University of Graz, and also by the FWF project P 28529-B27. Microscopic equipment is part of the Nikon-Center of Excellence, Graz that is supported by the Austrian infrastructure program 2013/2014, Nikon Austria Inc., and BioTechMed, Graz.
E.E, M.W., R.M., and W.F.G., staff members of the Medical University of Graz, have filed a U.K. patent application (patent application number WO2015EP74877 20151027, priority number GB20140019073 20141027) that describe parts of the research in this manuscript. Licenses related to this patent are provided to Next Generation Fluorescence Imaging (NGFI) GmbH (http://www.ngfi.eu/), a spin-off company of the Medical University of Graz.
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