Eroglu Emrah, Bischof Helmut, Charoensin Suphachai, Waldeck-Weiermaier Markus, Graier Wolfgang F, Malli Roland
Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.
Methods Mol Biol. 2018;1747:23-34. doi: 10.1007/978-1-4939-7695-9_3.
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.
一氧化氮（NO）是一种多功能信号分子，可调节生命所有领域的基本细胞过程。然而，由于NO的自由基性质，其半衰期非常短，这使得在单个细胞水平上追踪其形成、扩散和降解具有挑战性。最近，我们通过引入一类新型的基于单一荧光蛋白的NO探针——基因编码一氧化氮探针（geNOps），扩展了基因编码传感器家族。一旦在感兴趣的细胞中表达，geNOps会通过荧光猝灭对NO做出选择性反应，从而能够实时监测细胞内的NO信号。在这里，我们描述了适用于哺乳动物细胞中NO信号成像的详细方法。这种新方法可能有助于开展广泛的研究，以（重新）探究活细胞中复杂的NO生物化学过程。
