Reactivity of HYDROP-EX and HYDROP
The reactivity of HYDROP-EX is equivalent to that of HYDROP in cells, because cell-permeable HYDROP is hydrolyzed within cells to generate HYDROP-EX.
Reactivity of HYDROP-EX with reactive oxygen species
It shows high specificity to hydrogen peroxide (H2O2).
Reaction of HYDROP-EX with various reactive oxygen species.
Only H2O2 increases the fluorescence of HYDROP-EX at physiological pH of 7.4 or higher.
Fluorescence intensity of 10 µM HYDROP-EX was measured after addition of each 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 wavelength of 490 nm.
(Data was kindly provided by Prof. Dr. Y. Urano, Univ. Tokyo)
ROS generating system
H2O2: H2O2, 37℃, 60 min
・OH: Fe(ClO4)2:H2O2 =10:1, 37℃, 60 min
ONOO－: HOONO, 25℃, 5 min
ClO－: NaOCl, 25℃, 5 min
TBHP: tert-Butyl hydroperoxide
・NO: NOC13, 37℃, 30 min
1O2: EP-1, 37℃, 30 min
Reaction time course of HYDROP-EX with H2O2.
Fluorescence increase can be observed just after the addition of H2O2. Fluorescence intensity increases as the incubation time become longer.
10 μM of HYDROP-EX was dissolved to 0.1 M phosphate buffer (pH=7.4, including 0.1 % DMF as a cosolvent). H2O2 was added to each concentrations at the timepoint of 5 min (yellow arrow). Fluorescence intensity was measured using a microplate reader with excitation wavelength of 460 nm and emission wavelength of 520 nm at 37℃. Slit widths of excitation and emission was 9 nm and 20 nm, respectively.
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