mtC-geNOp is a cyan fluorescent genetically encoded sensor with a mitochondria targeting sequence capable for specific detection of nitric oxide (NO•) dynamics within mitochondria. It has been developed on the basis of cyan fluorescent protein (CFP) that is conjugated with a NO•-binding domain of the Escherichia coli protein NorR. Upon NO• binding to mtC-geNOp the cyan fluorescence intensity is immediately quenched. The NO•-dependent quenching effect correlates with the mitochondrial NO• concentration. Fluorescence of mtC-geNOp rapidly and fully recovers in the absence of NO• within mitochondria. Hence, mtC-geNOp provides a highly specific real-time read-out of mitochondrial NO• dynamics. The excitation and emission maxima of mtC-geNOp are at 430 nm and 480 nm, respectively. Standard optical filters optimized for imaging of CFP should be used.
mtC-geNOp is a genetically encoded cyan fluorescent biosensor that can be used to monitor NO• dynamics within mitochondria of intact living cells. In order to express mtC-geNOp in cells of interest 20 µg purified, endotoxin-free plasmid DNA coding for mtC-geNOp is provided. The plasmid coding for mtC-geNOp represents a mammalian expression vector with a strong viral promotor. We neither recommend using the plasmid as a source for the mtC-geNOp sequence nor multiplying it. 1 – 1.5 µg DNA should be used for cell transfection in a single well of a standard 6-well dish following standard transfection procedures. Usually cells express high amounts of mtC-geNOp 24 – 48 hours after cell transfection. In order to supply the NO•-binding domain with sufficient iron(II) cells need to be treated with the non-toxic iron(II) loading buffer for at least 10 minutes. To test the responsiveness of mtC-geNOp in cells, addition of potent NO-donors is recommended. High amounts of NO•-donors such as NOC-7 (10-100µM) should quench mtC-geNOp fluorescence by at least 10%. mtC-geNOp has been successfully used to visualize mitochondrial NO• dynamics in different cell types in response to various NO•-donors and is capable to detect endogenous Ca2+-triggered NO• production in endothelial and neuronal cells. To assess the NO•-selectivity of the mtC-geNOps signals a mutated probe, mtC-geNOpmut, is also available.