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NOFluor™ Series

DAF-FM DA (Diaminofluorescein-FM diacetate)

[For detection of intracellular nitrogen oxide by green fluorescence]

495-540 nm：Green

Diaminofluorescein-FM diacetate (DAF-FM DA) is a fluorescenct probe to detect intracellular nitric oxide (NO). DAF-FM DA is a cell permeable deacetylated form of DAF-FM, and  it is hydrolyzed by intracellular esterases to form cell impermeable DAF-FM, which reacts with NO. Compared with DAF-2 DA, it is improved and shows higher fluorescence at around pH 6.

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
SK1004-01 DAF-FM DA 1 mg

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  • Product Information

    DAF-FM DA (Diaminofluorescein-FM diacetate)
    Product Information

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    Principle of detection

    Cell permeable DAF-FM DA is hydrolyzed by intracellular esterases to generate cell impermeable and NO-reactive DAF-FM.
    Its fluorescence can be excited by blue light of ~495 nm in wavelength and emits green fluorescence of ~515 nm.

     

    Properties of DAF-FM

    Product Name target reaction pH range Absmax (nm) FLmax (nm) ε Φ
    DAF-FM NO irreversible >5.5 495 515 73,000 0.81

     

    Properties of the product

    Solution of DAF-FM DA 1 mg dissolved in 0.4 mL of DMSO.
    C25H18F2N2O7
    MW：496.42

FAQ

  • Q How long does it take for DAF-2 DA to be converted to DAF-2 by intracellular esterase?
    A

    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.

  • Q Can the fluorescence of DAFs be detected by a flowcytometer?
    A

    Yes, the application of DAF reagent measured by the flow site meter was reported by following papers.

    1. Havenga, M. J. E. et al. Simultaneous Detection of NOS-3 Protein Expression and Nitric Oxide Production Using a Flow Cytometer. Anal. Biochem. 2001, 290, 283-291.
    2. Navarro-Antolin, J. and Lamas, S. Nitrosative Stress by Cyclosporin A in the Endothelium: Studies with the NO-Sensitive Prove Diaminofluorescein-2/ diacetate using flow cytometry. Nephrol Dial Transplant, 2001, 16 [Suppl 1], 6-9.
    3. Jozsef L, Zouki C, Petasis NA, Serhan CN, Filep JG. Lipoxin A4 and aspirin-triggered 15-epi-lipoxin A4 inhibit peroxynitrite formation, NF-kappa B and AP-1 activation, and IL-8 gene expression in human leukocytes.  Proc Natl Acad Sci U S A 2002; 99: 13266-71.
  • Q Can I detect the fluorescence with a fixed-wavelength platereader of excitation at 485 nm and detection at 538 nm?
    A

    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.

     

  • Q What is the detection limit?
    A

    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.

  • Q Is there any cytotoxicity with these reagents?
    A

    The clear cytotoxicity was not recognized at around the concentration of 10 μM. In case the toxicity might be suspicious, please lower the concentration.

  • Q What is the optimum concentration?
    A

    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.

     

  • Q NO is a highly reactive compound, but, what state of NO does DAF series detect?
    A

    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.

  • Q How long DAF-2 DA, DAF-FM DA and DAR-4M AM retain inside the cells?
    A

    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.

  • Q Which of DAF/DAR reagents is the most appropriate for my purpose?
    A

    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

     

  • Q Any possibility to react with nitrite ion and nitrate ion ?
    A

    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.

  • Q Let me know example of use for these reagents.
    A

    There are many publications using the reagent. Refer the reference section or publication list. Cultured cell, vascular endothelial cell, cranial nerve system such as hippocampal, peripheral blood mononuclear cell, earthworm ganglion, and plant cell are used for test sample. Image observation with the fluorescent microscope, measuring the fluorescence intensity of the specific points of the image, and measurement with multi-well plate reader measurement.

     

  • Q Is there any compound interfering the reaction?
    A

    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.

  • Q What is recommended standard compound in case of determination by using calibration curve?
    A

    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.

  • Q What is the optimal loading time for incorporating reagents into cells.
    A

    There is no data for DAF series incorporating into cells. In the already reported examples, 1 hour to load DAF-2 DA into aortal smooth muscle cell of rats (Kojima, H. et al. Chem. Pharm. Bull, 1998, 46, 373-75), 30 minutes for DAR-4M AM loading into primary cultured endothelial cell (Kojima, H. et al. Anal. Chem. 2001, 73, 1967-73). Refer those published papers in the reference list and examine the optimal condition in your conditions.

  • Q Can DAF-FM DA be applied to bacteria to detect intracellular NOS?
    A

    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.

  • Q My question is not in this FAQ list.....
    A

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

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