NOFluor™ Series
[For detection of intracellular nitrogen oxide by green fluorescence]
495-540 nm：Green
Diaminofluorescein-2 diacetate (DAF-2 DA) is a fluorescent probe to detect intracellular nitrogen oxide (NO). This compound is cell permeable since it is diacetylated. It is hydrolyzed to form cell impermeable DAF-2 which retains inside the cells to detect intracellular NO.
| Code No. | Product Name | Size | Merck CAT No. |
Merck ( Millipore / Sigma Aldrich ) Product Name |
|---|---|---|---|---|
| SK1002-01 | DAF-2 DA | 1 mg |
DAF-2 DA (diaminofluorescein-2 diacetate)
Product Information
Cell permeable DAF-2 DA is hydrolysed by intracellular esterase to generate cell impermeable DAF-2.
DAF-2 reacts with NO to generate fluorescent DAF-2T, which can be excited with blue light (~495 nm) and emits green fluorescence (~515 nm).
| Product name | target | reaction | pH range |
Absmax (nm) | FLmax (nm) | ε | Φ |
| DAF-2 | NO | irreversible | >7 | 495 | 515 | 73,000 | 0.92 |
Solution of DAF-2 DA 1 mg dissolved in 0.45 ml of DMSO
C24H18N2O7
MW：446.4
It is difficult to measure the intracellular reaction kinetics directly, and there is no data. Preliminary experiment using homogenates from brain tissue showed that DAF-2 DA was converted within 10 minites.
Yes, the application of DAF reagent measured by the flow site meter was reported by following papers.
Yes. Excitation max at 495 nm and fluorescence max at 515 nm are the wavelength of each peaks. It can be measured at the wavelengths of slightly differs (Anal. Chem. 1998, 70, 2450). It is often observed that excitation at the wavelength shorter than the peak and detection at the wavelength longer than the peak lower the background and make better detection efficiency. Also refer the manual of the plate reader since it depends on the properties of the detector.
In the buffer of around pH 7 using DAF-2, NO detection limit is 5 nM. In case of using DAF-FM, it becomes slightly high sensitive as its intensity of fluorescence is 1.5 times higher than that of DAF-2. Lower sensitivity can be expected when DAF-2 DA or DAF-FM DA are used to detect intracellular NO because of intracellular interfering substances.
The clear cytotoxicity was not recognized at around the concentration of 10 μM. In case the toxicity might be suspicious, please lower the concentration.
Optimum concentration is about 5 – 10 μM (diluted by 500 – 1000 times). It could change by the kind of the sample and the buffer used. Due to the properties of the fluorescein-based compound, it may have an adverse effect if the density of the reagent is raised for getting a strong signal.
NO immediately reacts with oxygen, and gives NO2－ and NO3－. DAFs react with intermediates, which are generated from NO and oxygen. DAFs can detect NO specifically, because these intermediates cannot be produced without NO generation under physiological conditions.
DAF-2 DA, DAF-FM DA, and DAR-4M AM react cellular enzymes to generate cell DAF-2, DAF-FM, and DAR-4M, respectively. They are less cell permeable compared to original reagent but slowly leak outside the cells.
The first choice is DAF-FM for extracellular detection, and DAF-FM DA for intracellular detection.
DAF-2/DAF-2 DA is often used to reproduce the already reported results, because there are many publications using DAF-2 reagents. In contrast, DAF-FM/DAF-FM DA usually give better results at pH 6-7 range.
For the detection in a more acidic environment, or when green autofluorescence is not ignorable, DAR-4M/DAR-4M AM are good choices.
| Product code | Product name | Exmax (nm) | Emmax (nm) | Cell permeability | Capable pH range |
| SK1001-01 | DAF-2 | 495 | 515 | – | >7 |
| SK1002-01 | DAF-2 DA | 495 | 515 | + | >7 |
| SK1003-01 | DAF-FM | 495 | 515 | – | >5.5 |
| SK1004-01 | DAF-FM DA | 495 | 515 | + | >5.5 |
| SK1005-01 | DAR-4M | 560 | 575 | – | 4-12 |
| SK1006-01 | DAR-4M AM | 560 | 575 | + | 4-12 |
DAF series will not react with nitrite ion (NO2－)and nitrate ion (NO3－) under physiological conditions. However, if they are incubated for a long time in the presence of high concentration NO2－ (≧ 10 mM), slight fluorescence will be observed.
Fluorescent substances such as phenol red and vitamins sometimes interfere the observation. Proteins such as serum and BSA added to medium also sometimes lower the sensitivity of NO detection.
For the determination of absolute concentration, it is necessary to use the NO gas solution of known density. However, practically, NONOate (NO of 2 molecules is generated by 1 molecule) is recommended to use.
Many bacteria secret various proteases including esterases to the extracellular fluid, and thus DAF-FM DA may be hydrolyzed before entering into the cells. For example, it has been reported that DAF-FM DA was hydrolyzed before entering into B. subtilis and did not incorporated to the cells. In general, diacetylated fluorescent probes are used for eukaryotic cells, and may be difficult to be used for prokaryotes.
Please also refer the frequently asked questions on fluorescent probes
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