Need a simple mathematical solution to quantify the NO in side the cells using DAF-FM as NO detector. I have used one NO delivering agent to increase the NO in cells but could not able to quantify. Already I got significant increased fluorescent intensity using DAF-FM due to treatment of our NO delivery agent showing NO release but need the quantification (uM?).
Maybe possible to record fluorescent signal from cell exposed to NO relising compounds with known rate. But problem of differetiation of intra/extracellular NO will not disapeared.
An option: Stimulate any macrophages with different concentration of LPS and record DAF-FM signal 6-8 hr later. In parallel to this estimate NOx- production for 2 hr and compare data. In vitro system without cells but with NO donor with known rate also could help to validate results.
Good luck!
There is a more sophisticated aproach by using a newly developped genetically encoded NO sensor called geNOps by a company called Next Generation FLuorescence Imaging (NGFI). Please check the paper (Eroglu et al. Development of novel FP-based probes for live-cell imaging of nitric oxide dynamics. Nat. Commun. 7: 10623, 2016) and the suppliers website: www.ngfi.eu. Perhaps you may find this sensor very usful as its very easy to use and excellent in reproductivity and sensitivity. The company also offers "sensory cells" so you can avoid transfection of your cells if this is a problem.
If not able to quantify, then you can always still report it as a fold increase compared to control by adjusting the control fluorescence to 100%.
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