ROSFluor™ Series


[Nitrative stress sensing probe]

495-540 nm:Green

NiSPY-3 (Nitrative Stress Sensing Pyrromethene Dye) is a reagent to detect nitrosative stress by fluorescence. NiSPY-3 reacts specifically with peroxynitrite (ONOO) among other reactive oxygen species such as ・OH, ・O2 , H2O2, 1O2, NO, etc. Fluorescent intensity is not increased by the existence of hydroxyl radical, singlet oxygen, hydrogen peroxide, hypochlorite, nitric oxide or superoxide. It can beused for live cell fluorescent imaging.


Available through Merck KGaA (Darmstadt, Germany) as:
SCT035 BioTracker™ 515 Green ONOO- Dye


Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
SK3003-01 NiSPY-3 1 mg SCT035 BioTracker 515 Green ONOO- Dye


  • Protocol

  • SDS

  • Product Information


    Principle of detection with NiSPY-3

    NiSPY-3 does not show fluorescence in neutral solutions. When NiSPY-3 reacts with peroxynitrite, it becomes a strongly fluorescent substance (excitation: 490 nm, emission: 515 nm).



    NiSPY-3  1 mg
    C23H19BF2N4O4   Mw: 464.23

    Preparation of the reagent

    Dissolve all the reagent in 430 μL of DMSO (5mmol/L), and then dilute 500-5000 times with neutral buffer (final concentration: 1-10 μM) before use. We recommend to use up at once after probes are diluted.


    Reprinted in part with permission from (J. Am. Chem. Soc. 2006,  128, 33, 10640-10641).  Copyright (2006) American Chemical Society.

    A)Fluorescence spectra of NiSPY-3 solution (10 μM NiSPY-3 in 0.1 M phosphate buffer pH 7.4  containing 0.1% DMF as a cosolvent) upon addition of peroxynitrite (final 0, 1, 2, 5,10, 20 μM).

    B)Fluorescence response of NiSPY-3 in various ROS generation systems



  • Q Let me know an example protocol for live-cell imaging.

    This protocol is for the staining of HeLa cells. Add 2 μM NiSPY-3 to the culture medium (DMEM + 8% FBS + 100 units/mL penicillin + 100 mg/mL streptomycin) with 0.01% DMF and 0.01% Cremophor EL as cosolvent, and culture for 20 minutes. Then wash the cells with HBSS 2 times and observe by fluorescent microscopy. Use blue excitation filter set or 488 nm laser excitation. Post stimulation by peroxynitrite may increase the intracellular peroxynitrite concentration and increase in the fluorescence of NiSPY-3.


G. Uchida, Y. Mino, T. Suzuki, J. Ikeda, T. Suzuki, K. Takenaka Y. Setsuhara (2019)
Sci Rep. 9:6625 DOI: 10.1038/s41598-019-42959-4.

Y. Kimura, K. Takashima, S. Sasaki, T. Kaneko (2018)
J. Phys. D: Appl. Phys. 52: 064003, DOI: 10.1088/1361-6463/aaf15a

R. Furuta, N. Kurake, K. Ishikawa, K. Takeda, H. Hashizume, H. Tanaka, H. Kondo, M. Sekine, M. Hori (2017)
Plasma Process. Polym. 14 1700123 DOI:10.1002/ppap.201700123

T. Ueno, Y. Urano, H. Kojima, T. Nagano (2006)
J. Am. Chem. Soc. 128: 10640-10641 DOI:10.1021/ja061972v