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NOFluor™ Series

DAR-4M AM (diaminorhodamine-4M acetoxymethyl ester)

[For detection of intracellular nitrogen oxide by orange fluorescence]

570-590 nm：Orange

DAR-4M AM is a fluorescent probe to detect intracellular nitrogen oxide (NO). This compound is cell permeable since it is an acetoxymethyl ester form of DAR-4M.  It becomes into DAR-4M by intracellular esterases and it retains inside the cells. 

Compared to DAF-2 DA or DAF-FM, it can be used in wider range of pH (4-12).

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
SK1006-01 DAR-4M AM 1 mg

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    DAR-4M AM (diaminorhodamine-4M acetoxymethyl ester)
    Product Information

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    Principle of detection

    Cell permeable DAR-4M AM is hydrolyzed by intracellular esterase to generate cell impermeable and NO-reactive DAR-4M.
    Its fluorescence signal can be excited by green light of ~560 nm in wavelength to emit orange fluorescence of ~575 nm in wavelength.

    Properties of DAR-4M

    Product Name
    		 target reaction pH range Absmax (nm) FLmax (nm) ε Φ
    DAR-4M NO irreversible 4-12 560 575 76,000 0.42

    Spectra

    Properties of the product

    Solution of 5 mmol/L. Dilute ~500 times with neutral aqueous bugger solutions.

     

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

    Please also refer the frequently asked questions on fluorescent probes

     

Reference

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M. Satoh, S. Fujimoto, Y. Haruna, S. Arakawa, H. Horike, N. Komai, T. Sasaki, K. Tsujioka, H. Makino, N. Kashihara (2005)
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H. Kojima, M. Hirotani, N. Nakatsubo, K. Kikuchi, Y. Urano, T. Higuchi, Y. Hirata, Nagano, T (2001)
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R. Furuuchi, I. Shimizu, Y. Yoshida, Y. Hayashi, R. Ikegami, M. Suda, G. Katsuumi, T. Wakasugi, M. Nakao, T. Minamino (2018)
PLoS One 13: e0202051 DOI:10.1371/journal.pone.0202051

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