ΔO-geNOp is a orange fluorescent genetically encoded negative control that does not respond to intracellular nitric oxide (NO•) dynamics. It has been developed on the basis of the orange fluorescent protein (OFP) that is conjugated with a mutated domain of the Escherichia coli protein NorR. In the presence of NO• the orange fluorescence intensity of ΔO-geNOp is not affected. Hence, ΔO-geNOp should be used for control experiments. The excitation and emission maxima of ΔO-geNOp are at 550 nm and 565 nm, respectively. Standard optical filters optimized for imaging of OFP should be used.
ΔO-geNOp is a genetically encoded orange fluorescent construct that can be used as a negative control. In order to express ΔO-geNOp in cells of interest 20 µg purified, endotoxin-free plasmid DNA coding for ΔO-geNOp is provided. The plasmid coding for ΔO-geNOp represents a mammalian expression vector with a strong viral promotor. We neither recommend using the plasmid as a source for the ΔO-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 ΔO-geNOp 24 – 48 hours after cell transfection. Even high amounts of NO•-donors such as NOC-7 (10-100µM) should not quench ΔO-geNOp fluorescence.
Main Steps for correct ΔO-geNOp usage and data interpretation:
- Transfect cells with 1-1.5 µg/ml plasmid DNA coding for ΔO-geNOp
- 24-48h after transfection keep cells for 10 min in the iron(II) loading buffer prior to fluorescence microscopy
- Wash cells and image orange fluorescence of ΔO-geNOp expressing cells over time using fluorescence microscopy
- Perform your protocols of interest. Changes of ΔO-geNOp fluorescence are not due cytosolic NO• fluctuations
- The NO• sensitive O-geNOp should be used under the same experimental conditions
Eroglu et. al. Nat. Comm. 2016