Updated: 25 September 2019
For research use onlyĂ

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Table 1. Product information

1.

Catalog no.

Material

AmountStorage upon receipt

Stability

GC3007-01

HYDROP

30 nmol × 3

GC3008-01

HYDROP-EX

30 nmol × 3≤−20°C, keep
desiccated andprotected from light.

1 year (whenunopenedand stored asdescribed.)

About HYDROP and HYDROP-EX

HYDROP and HYDROP-EX are fluorescent probes tospecifically detect hydrogen peroxide (H2O2), one of thereactive oxygen species (ROS). These are initially nonfluorescent but fluoresce upon reaction with hydrogenperoxide in physiological conditions.
HYDROP is a probe to specifically detect intracellular
H2O2. It is diacetylated and highly cell-permeable, but not̀

reactive to H2O2 before entering into cells. After hydrolysis
of diacetyl groups by intracellular esterases, it becomes
highly reactive to H2O2.
Cell impermeable HYDROP-EX is suitable to detect orquantify extracellular H2O2 or that in solutions

̀■ Storage
Probes are shipped at room temperature in a nitrogen gasfilled vial. Upon receipt, store the product desiccated andprotected from light at ≤ −20°C. Storing the reagent as asolution is not recommended.

2.
Prepare N,N-dimethylformamide (DMF) as a solvent.
HYDROP is a colorless solid, HYDROP-EX is anorange-colored solid. Before opening the cap, warm the vial
to the room temperature and use micro-centrifuge to spin
down the solid that might be adhered on the cap. Add 30
̀L of DMF to one vial to prepare 1 mM solution. Dissolve
the solid completely by pipetting for more than five times.
HYDROP solution will be colorless, whereas HYDROP-EX
solution will be orange.

3.
1. Prepare hydrogen peroxide (H2O2) solutions togenerate a standard curve. Commercially available
30−35% H2O2 solution is about 10 M. Dilute the solution
1000 times with pure water and measure absorbance at
240 nm (A240).
2. Concentration of the H2O2 can be obtained by theequation: C = A240 / ε × dilution factor (M), whereas ε =43.6 (M-1cm-1) is the molar extinction coefficient of H2O2.
3. Dilute the H2O2 to the final concentration of 0−100 ̀Musing a solution. The diluting solution condition shouldbe equivalent to that in which you measure H2O2concentrations. Add HYDROP-EX to final concentrationof 1−10 ̀M, react at 37oC for 1 hour. These
concentrations and reaction conditions should beadjusted to the expected conditions to be measured.
4. Measure fluorescence intensities using a fluorescence
spectrometer or a fluorescence microplate reader.
Before the measurement, dilute the solution to make thefinal HYDROP-EX concentration to be <1 ̀M, orabsorbance of the solution at 490 nm should be lessthan 0.1. We recommend to set the excitation
wavelength within 470−490 nm and emission
wavelength to be 520−530 nm.
5. Plot the measured fluorescence intensity (I) against
H2O2 concentration (C) and fit using the following
equation,to obtain the parameters, a, b, and k.

Fig. 2. An example standard curve of HYDROP-EX.

4.
1. Dilute the 1 mM HYDROP solution with observationbuffer or culture media to 1−5 µM (Cell stainingsolution).
※°C°C

2. Remove the culture medium on the dish and wash twicewith the observation buffer or the culture medium.

3. Add the cell staining solution to the dish and incubate at37°C for 20 min.

4. After the staining, wash 2 times with the observationbuffer.

5. Induce the production ofby theaddition of PMA and start※

6. Add the same concentration of HYDROP-EX to thesolution you would like to measure, mix well andincubate in the same conditions (temperature and time).
After the incubation, measure fluorescence.
7. Obtain H2O2 concentrations using the followingequation,■ Fluorescence observation
Use 488 nm blue light for excitation.
516 nm. For fluorescence microscopy, blueexcitation filter sets for GFP or FITC is appropriate.

Table 2. Related Products
Catalog no.

Material

Usage

SK3001-1
SK3001-2

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Specific fluorescence probe to detect hydroxyl radical ( ・ OH) andperoxynitrite (ONOO−).

SK3002-01
SK3002-02

APF

Specific fluorescence probe to detect hydroxyl radical (・OH), peroxynitrite(ONOO−) and hypochlorous acid (HClO).

SK3003-01

NiSPY-3

Specific fluorescence probe to detect only peroxynitrite (ONOO−).

GC3006-01

HySOx

Specific fluorescence probe to detect only hypochlorous acid (HClO).

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AcidiFluor™ ORANGE

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FeRhoNoxTM-1

A fluorescence probe to detect ferrous

A401-1

QuicGSH3.0Quantification of intracellular glutathione.

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