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[Measurement of intracellular reactive oxygen species(ROS)]HYDROP™

  • High specificity to hydrogen peroxide. 
  • It is suitable for time-lapse imaging because it photobleaches slowly.


HYDROP™ is a fluorescent probe which is non-fluorescent under physiological condition, but fluoresces upon reaction with hydrogen peroxide (H2O2). Hydrogen peroxide is one of the reactive oxygen species (ROS) including hydroxyradical (OH), superoxide (O2-・), hypochlorous acid (OCl), singlet oxygen (1O2), nitric oxide (NO), and peroxynitrite (ONOO). HYDROP™ shows high selectivity towards hydrogen peroxide compared to other ROS (OH, O2-・,ClO, 1O2, NO, ONOO).

Principle of the measurement

Cell permeable HYDROP™ hydrolyzed by intracellular esterases to yield a material that is impermeable to the cell membrane and thus stays within the cell. The hydrolysis also increases the reactivity with hydrogen peroxide. HYDROP and its hydrolyzed product are almost non-fluorescent in neutral solutions. After the reaction with hydrogen peroxide, it generates a highly fluorescent product (excitation max: 492 nm, emission max: 518 nm).

ROS Selectivity

Figure 1. Reaction of HYDROP to various ROS
Figure 1. Reaction of HYDROP™ to various ROS

Fluorescence increase of hydrolyzed HYDROP™ is observed by the addition of hydrogen peroxide at physiological pH of around 7.4 or higher.

  • Fluorescence intensity of 10 µM hydrolyzed HYDROP™ after addition of various ROS (final conc. 50 µM) in 0.1 M sodium phosphate buffer at pH 7.4 containing 0.1 % DMF as a cosolvent.
  • Fluorescence intensities were measured at 520 nm, with excitation at 490 nm.
ROS generating system

O2-・: KO2
H2O2: H2O2, 37℃, 60 min
OH: Fe(ClO4)2 : H2O2 =10:1, 37℃, 60 min
ONOO: ONOO, 25℃, 5 min
ClO: NaOCl, 25℃, 5 min
TBHP: tert-Butyl hydroperoxide
NO: NOC13, 37℃, 30 min
1O2: EP-1, 37℃, 30 min


Figure 2. Spectra

Fluorescence intensity of 5 µM HYDROP™ after addition of 50 µM hydrogen peroxide in 0.1 M sodium phosphate buffer at pH 7.4 containing 0.1 % DMF as a cosolvent.

  • Absorbance intensities were measured with slit width 1.5 nm using HITACHI U-2910 Spectrophotometer.

  • Fluorescence intensities were measured 518nmwith excitation at 492 nm, slit width 2.5 mm, photon multiplier voltage 700 V, using HITACHI F-2700 Fluorescence Spectrophotometer.

Visualization of hydrogen peroxide production by RAW 264.7 cells (1×105 cell/ml) using HYDROP™. Green fluorescent signal is overlayed to DIC image. Hydrogen peroxide production was induced by the addition of 0.001 µg/ml phorbol myristate acetate (PMA). Cells were stained with 1 µM HYDROP™ for 20 min. Bar, 25 µm.

Please refer to the application note

Kazuo Tomita, Yoshikazu Kuwahara, Yuko Takashi, Takao Tsukahara, Akihiro Kurimasa, Manabu Fukumoto, Yoshihiro Nishitani, Tomoaki Sato. (2017)
Biochem. Biophys. Res. Commun. 490: 330-335  DOI: 10.1016/j.bbrc.2017.06.044
Kolanowski JL, Kaur A, New EJ. (2016)
Antioxid Redox Signal. 24:713-30
Guo H, Aleyasin H, Dickinson BC, Haskew-Layton RE, Ratan RR (2014)
Cell Biosci. 4:64
Masahiro Abo, Yasuteru Urano, Kenjiro Hanaoka, Takuya Terai, Toru Komatsu, and Tetsuo Nagano
J. Am. Chem. Soc., 2011, 133 (27), pp 10629–10637 doi: 10.1021/ja203521e
Code No. Product Size Price Protocol MSDS
GC3007-01 HYDROP™ 30nmolx3 $398 Download