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[Measurement of highly reactive oxygen species (hROS) by orange fluorescence] OxiORANGE™

OxiORANGE™ is an orange fluorescent probe to detect hydroxy radical (OH) or hypochlorous acid (HClO) in live-cell imaging. Its nearly red fluorescence spectrum allows multicolor imaging with green (ex. GFP, FITC) and blue (ex. Hoechst 33342) fluorophores.
Because of its positive charge, OxiORANGE™ tends to localize within mitochondria. It has high photostability and is suitable for time-lapse imaging of intracellular hROS generation.

Features

  • It can detect hydroxy radical (OH) and hypochlorous acid (HClO) among reactive oxygen species (OH, O2-・,HClO, H2O2, NO, ONOO).
  • Suitable for live-cell imaging or time-lapse imaging because of its bright fluorescence with high photostability.
  • Capability of multicolor imaging: It can be used with green and blue fluorophores such as GFP and Hoechst 33342.
  • It tends to localize within mitochondria and it shows stable localization.
  • Fluorescence is stable after mild fixation (3-4 % PFA, 20 min).

Principle of the measurement

OxiORANGE™ is almost non-fluorescent in neutral buffer solutions. It fluoresces by the reaction with hydroxy radical (OH) or hypochlorous acid (HClO). Its absorbance maximum is 553 nm, and emission maximum is 577 nm (orange fluorescence). It penetrates through cell membrane and localizes in mitochondria by the membrane potential.  

Fluorescence and reactivity

Fluorescence and reactivity

Absorbance/fluorescence spectra (left) and reactivity with various ROS (right).
About 30 times fluorescence increase after reaction with hydroxy radical (・OH) is observed.

Measurement conditions for the spectra.
  • Absorbance and fluorescence spectra of 10 μM of OxiORANGE™ dissolved in phosphate buffer (0.1M, pH 7.4 with 0.1% DMF as a cosolvent) were measured after addition of 5 µM of NaOCl.
  • OxiORANGE™ was excited by 553 nm light, with slit widths of 2.5 nm and fluorescence measured at 577nm with photon multiplier voltage of 700V.
Measurement conditions for the reactivity.
  • 10 μM of OxiORANGE™ was dissolved to phosphate buffer (0.1M, pH 7.4 with 0.1% DMF as a cosolvent). Then the following reagents was added to generate reactive oxygen species.
  • Fluorescence at 577 nm was measured by the excitation at 553 nm, with a slit width of 2.5 nm and photon multiplier voltage of 700V.
ROS generating system

OH: 300 µM Fe(ClO4)2 , 1 mM H2O2 , RT, 5 min
ONOO: ONOO 50 µM, RT, 5 min
HClO: NaOCl 50 µM, RT, 5 min
NO: NOC18 5 µM, 37℃, 30 min
O2: KO2 100 µM, 37℃, 30 min
H2O2: H2O2 100 µM, 37℃, 30 min

Features 1: Bright and stable fluorescence

Bright and stable fluorescence 

Comparison between mitochondria-localizing probes to detect oxidative stress. RBL-2H3 cells loaded with 1 μM of OxiORANGE™ (above), product A (center), or product B (bottom) were stimulated by the addition of 0.5 μM H2O2. Photos were taken just after the addition of the probes (left) and 20 minutes later (right) in the same excitation/observation conditions. OxiORANGE™ shows the brightest fluorescence among these products. Product B migrated into nucleus. In contrast, localization of OxiORANGE™ was stable.

Images were taken using NIKON ECLIPSE Ti, (PlanFluor 40×0.75), and Hamamatsu ORCA-R2 camera.

Comparison with a ROS-detecting probe.

Comparison with a ROS-detecting probe. OxiORANGE™ (1 μM, top, orange) or other product C (5 μM, bottom, deep red) was added to the medium and incubated for 30 minutes. After the medium was exchanged to HBSS, 1 mM H2O2 was added to stimulate ROS production. Bright signal from OxiORANGE™ was detected. DIC image (gray),Hoechst 33342 (blue), and OxiORANGE™ (orange), or Product C (red) is overlaid.
Images were taken using Leica DMI6000 CS (HC PL Apo 40×0.85).

Features 2: Fluorescence can be observed after mild fixation.

OxiORANGE™ fluoresces after reaction with ROS. The reaction is irreversible and the fluorescence remains after mild fixation with 3-4% PFA for 5-20 minutes.

 

Fluorescence can be observed after mild fixation.

Fluorescence of OxiORANGE™ before and after the fixation. HeLa cells were cultured for 30 minutes in the presence of 1 μM of OxiORANGE™ and 0.2 μg/mL Hoechst 33342. Cells were rinsed with HBSS two times, stimulated with 500 μM of H2O2, then ROS generation was observed after 30 minutes (left). Next, cells were fixed with 3% PFA containing PBS (pH 7.4) at 4℃ for 20 minutes. Cells were observed in the same condition. Images of red: OxiORANGE™, blue: Hoechist33342, and gray: DIC are overlaid.
※ Images were taken using NIKON ECLIPSE Ti, (PlanFluor 40×0.75) ), and Hamamatsu ORCA-R2 camera.

Localization of OxiORANGE can be changed by the fixation. Fluorescence intensity might be slightly decreased by the fixation. Addition detergent for the cell permeabilization may decrease the fluorescence. Please test the fixation conditions prior to your experiments.

Localization of OxiORANGE™ within cells.

OxiORANGE tends to localize within mitochondria if the concentration of the reagent is low enough. OxiORANGE™ also distribute to some other parts of cells, especially when the concentration of OxiORANGE™ is higher, or when other mitochondria-localizing reagents was added.

Localization of OxiORANGE™ within cells.

  • HeLa cells were stained with 0.5 μM of OxiORANGE™, 0.25 μM of MitoTrackerGREEN, and 0.2 μg/mL of Hoechst33342 for 30 minutes.
  • Stimulated with 100 μM of hydrogen peroxide, for 30 min.
  • Observed by fluorescence microscopy.

See also application note for the detailed protocols.

Reference
Yuichiro Koide, Yasuteru Urano, Suguru Kenmoku, Hirotatsu Kojima, and Tetsuo Nagano (2007)
Design and Synthesis of Fluorescent Probes for Selective Detection of Highly Reactive Oxygen Species in Mitochondria of Living Cells
J. Am. Chem. Soc., 129(34) 10324–10325 doi: 10.1021/ja073220m
Price
Code No. Product Size Price Protocol MSDS
GC3004-01 OxiORANGE™ 100 nmol ×5 $498  width=
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E-mail: info_itnl@goryochemical.com