ΔC-geNOp is a cyan fluorescent genetically encoded negative control that does not respond to intracellular nitric oxide (NO•) dynamics. It has been developed on the basis of cyan fluorescent protein (CFP) that is conjugated with a mutated domain of the Escherichia coli protein NorR. In the presence of NO• the cyan fluorescence intensity of ΔC-geNOp is not affected. Hence, ΔC-geNOp should be used for control experiments. The excitation and emission maxima of ΔC-geNOp are at 430 nm and 480 nm, respectively. Standard optical filters optimized for imaging of CFP should be used.
ΔC-geNOp is a genetically encoded cyan fluorescent construct that can be used as a negative control. In order to express ΔC-geNOp in cells of interest 20 µg purified, endotoxin-free plasmid DNA coding for ΔC-geNOp is provided. The plasmid coding for ΔC-geNOp represents a mammalian expression vector with a strong viral promotor. We neither recommend using the plasmid as a source for the ΔC-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 ΔC-geNOp 24 – 48 hours after cell transfection. Even high amounts of NO•-donors such as NOC-7 (10-100µM) should not quench ΔC-geNOp fluorescence.
Main Steps for correct ΔC-geNOp usage and data interpretation:
- Transfect cells with 1-1.5 µg/ml plasmid DNA coding for ΔC-geNOp
- 24-48h after transfection keep cells for 10 min in the iron(II) loading buffer prior to fluorescence microscopy
- Wash cells and image cyan fluorescence of ΔC-geNOp expressing cells over time using fluorescence microscopy
- Perform your protocols of interest. Changes of ΔC-geNOp fluorescence are not due cytosolic NO• fluctuations
- The NO• sensitive C-geNOp should be used under the same experimental conditions
Eroglu et. al. Nat. Comm. 2016