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ROSFluor™ Series

OxiORANGE™

[Reactive oxigen species (ROS) detecting probe]

570-590 nm：Orange

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.

 

Available through Merck KGaA (Darmstadt, Germany) as:
SCT 038 BioTracker™ Orange OH and HCIO Dye

 

 

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC3004-01 OxiORANGE™ 100 nmol ×5 SCT038 BioTracker Orange OH and HCIO Dye

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  • Product Information

    OxiORANGE™
    Product Information

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    Properties of OxiORANGE

    Product Name Target Cell permeability Reacivity  Absmax (nm)  FLmax (nm)
    OxiORANGE ・OH, HClO Yes irreversible 553 577

     

    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 detection

    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.

    Spectra and reactivity

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

    Measurement conditions for the spectra.
    • Absorbance and fluorescence spectra of 10 μM of OxiORANGE dissolved in phosphate buffer (0.1 M, 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 577 nm with photon multiplier voltage of 700 V.

     

    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 700 V.
    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

  • Cell imaging examples using OxiORANGE

    OxiORANGE™
    Cell imaging examples using OxiORANGE

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    Cell imaging examples using OxiORANGE

    Features 1: 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. 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 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.

    HeLa cells were loaded with 0.5 μM of OxiORANGE, 0.25 μM of MitoTrackerGREEN, and 0.2 μg/mL of Hoechst33342, then stimulated with 100 μM of hydrogen peroxide, for 30 min.

  • Detection of hROS generation in HeLa cells using OxiORANGE

    OxiORANGE™
    Detection of hROS generation in HeLa cells using OxiORANGE

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    Detection of hROS generation in HeLa cells using OxiORANGE

    Experiment procedure

    1. Added 100 μL of dimethylformamide (DMF) to one vial of OxiORANGE (100 nmol) to prepare 1 mM solution.
    2. Diluted the 1 mM solution with cell culture medium to make 1 µM reacting solution.
    3. Removed the cell culture medium, rinsed with HBSS, and replaced the medium with the reacting solution described above. Incubated for 20 minutes at 37℃, 5% CO2
    4. Rinsed the cells with HBSS twice and replaced the medium with cell culture medium without phenol red. Place the cells in a CO2 incubator chamber on a microscope for observation.
    5. Added final 500 μM of H2O2 to the medium to induce intracellular hROS production and simultaneously, start time-lapse imaging.
    1.  

    ※ Optimum reagent concentrations and reaction time might be varied depending on the cell culture conditions and cell species. In our lab., staining with 1 µM solution for 20 minutes at 37℃ gave good results for HeLa, RAW264.7, RBL-2H3, and HL–60 cells.
    ※ Since it has been reported that in a starved condition ROS production in HeLa cells was induced,  we recommend to use cell culture medium instead of buffers such as PBS or HBSS. For microscopy, we recommend to use cell culture medium without phenol red because phenol red shows fluorescence.

     

    hROS generation detected with OxiORANGE in HeLa cells.
    Cells were observed using NIKON ECLIPSE Ti, microscope (PlanFluor 40×0.75), and Hamamatsu ORCA-R2 camera.  Addition of antioxidant (N-acetyl-cysteine, NAC) reduces ROS production (right).

     

    Youtube timelapse video.

  • A timecourse of ferroptosis observed by fluorescence live cell imaging using FerroOrange and ROSFluor series

    OxiORANGE™
    A timecourse of ferroptosis observed by fluorescence live cell imaging using FerroOrange and ROSFluor series

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    A timecourse of ferroptosis observed by fluorescence live cell imaging using FerroOrange and ROSFluor series

    Ferroptosis is a cell death which depends on intracellular labile Fe2+. Its mechanism is known as a distinct from that of apoptosis or necrosis. Excess amount of intracellular labile Fe2+ induces generation of reactive oxigen species (ROS) and lipid peroxidation, those lead to cell death. It has also been revealed that ferroptosis is induced in some neurodegerative diseases, and some tumor cells are ferroptosis resistant.

    Here, we tried to detect labile Fe2+ that induces ferroptosis, as well as ROS, by using activatable fluorescent probes of FerroOrange, APF, OxiORANGEand HYDROP, in the timecourse of ferroptosis.

    Visualization of Fe2+ and ROS in the process of ferroptosis

    Erastin was applied to HT-1080 cells to induce ferroptosis, and intracellular labile Fe2+ and ROS were imaged after 3, 6, 9 hours after the application, using the fluorescent probes. FerroOrange (1 μM),  APF (5 μM), HYDROP (1 μM), and OxiORANGE (1 μM) were applied  30 minutes before each of the observation timepoint. Fluorescence signal of FerroOrange which indicates labile Fe2+ was maximum at 3 hours after the induction, whereas fluorescence of other ROS probes: APF which detects hydroxyradical (·OH), hypochlorous acid (HClO), and peroxinitrite (ONOO–), OxiORANGE which detects hydroxyradical (·OH), hypochlorous acid (HClO), and HYDROP which specifically detects hydrogen peroxide H2O2 were maximum at 6 hours after the induction. These results indicate that in ferroptosis, ROS increase after the increase in labile Fe2+.

    Fluorescence intensity changes of FerroOrange and APF in ferroptosis-induced HT-1080 cells

    Fluorescence intensity of FerroOrange and APF in erastin-applied HT-1080 cells (upper and middle rows) were shown. Images were overlayed as pseudocolor images (bottom). Magenta indicates fluorescence of FerroOrange, green indicates that of APF. Bar indicates 100 µm.

    Fluorescence intensity changes of OxiORANGE and HYDROP in ferroptosis-induced HT-1080 cells

    Fluorescence intensity of OxiOrange and HYDROP in erastin-applied HT-1080 cells (upper and middle rows) were shown. Images were overlayed as pseudocolor images (bottom). Magenta indicates fluorescence of OxiOrange, green indicates that of HYDROP. Bar indicates 100 µm.

     

    Experiment procedure

    1. HT-1080 cells of 2 × 105 were seeded on glass bottom dishes and cultured until the cells attached to the bottom.
    2. Erastin (final concentration of 30 μM) was added to the media and cultured for2.5, 5.5, 8.5 hours at 37℃, 5% CO2.
    3. Each probes were added to the media and cultured for 30 minutes.
    4. The cells were rinsed twice with HBSS and observed by fluorescence microscopy.

    ※In different cell culture conditions, the concentrations of the reagents and incubation time might be varied. In this experiment, preliminary tests were needed to optimize those conditions.
    ※If the cell adhesion to the dishes was weak, we recommend to use poly-L-lysine coated dished.

    Actual timecourse of the experiment. Actually, erastin was added in different timepoints and addition of fluorescent probes and observations were performed simultaneously. Since these probes reacts with each target and fluoresces irreversibly, timecourse of ferroptosis was observed by the above procedure.

     

FAQ

  • Q Can I use DMSO for dissolving OxiORANGE, HYDROP, APF, HPF?
    A

    We recommend to use DMF for these reagents because DMSO is known to be a quencher of one of the reactive oxygen species (ROS), hydroxy radicals. Even though you are intended to detect aother ROS, the ROS could be generated by the signal of hydroxy radicals and usage of DMSO could also decrease the target ROS. Therefore we recommend to use DMF instead of DMSO.

     

  • Q Tell me a selection guide of ROSFluor series.
    A
    Product code Product name Exmax (nm) Emmax (nm) ・OH ONOO- HClO H2O2 O2-・ ROS detection in solutions ROS detection in cells
    GC3004-01 OxiORANGE 553 577 + － + － － + +
    GC3006-01 HySOx 553 574 － － + － － + +
    GC3007-01 HYDROP 492 518 － － － + － – +
    GC3008-01 HYDROP-EX※ 492 518 － － － + － + –
    SK3001-01 HPF 490 515 + + － － － + +
    SK3002-01 APF 490 515 + + + － － + +
    SK3003-01 NiSPY-3 490 515 － + － － － + +

    ※HYDROP-EX is suitable for detection of extracellular H2O2 since it has low cell permeability.

Reference

Y. Takenaka, I. Inoue, T. Nakano, M. Ikeda, Y. Kakinuma (2022)
FEBS J. 289: 1650-1667 DOI: 10.1111/febs.16249

E. Nakayama, T. Kushibiki, Y. Mayumi, R.i Azuma, M. Ishihara, T. Kiyosawa (2022)
Biology 11: 301 DOI: 10.3390/biology11020301

Y. Okina, M. Sato-Matsubara, T. Matsubara, A. Daikoku, L. Longato, K. Rombouts, L. T. T. Thuy, H. Ichikawa, Y. Minamiyama, M. Kadota, H. Fujii, M. Enomoto, K. Ikeda, K. Yoshizato, M. Pinzani, N. Kawada (2020)
J. Hepatol. 73: 882 DOI: 10.1016/j.jhep.2020.03.051

A. Sakasai-Sakai, T. Takata, M. Takeuchi (2020)
Int. J. Mol. Sci. 21: 4861  DOI:10.3390/ijms21144861

F. Yagishita, J. Tanigawa, C. Nii, A. Tabata, H. Nagamune, H. Takanari, Y. Imada, Y. Kawamura (2019)
ACS Med. Chem. Lett. 10: 1110

K. Marunaka, M. Kobayashi, S. Shu, T. Matsunaga, A. Ikari (2019)
Int. J. Mol. Sci. 20: 3869 DOI: 10.3390/ijms20163869

F. Sugimori, H. Hirakawa, A. Tsutsui, H. Yamaji, S. Komaru, M. Takasaki, T. Iwamatsu, T. Uemura 2, Y. Uemura, K. Morita, T. Tsumura. (2019)
PLoS One 14: e0213579. DOI: 10.1371/journal.pone.0213579.

Y. Koide, Y. Urano, S. Kenmoku, H. Kojima, T. Nagano (2007)
J. Am. Chem. Soc. 129: 10324-10325 DOI:10.1021/ja073220m

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