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.