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ROSFluor™ Series

HySOx

[For the specific detection of hypochlorous acid]

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570-590 nm：Orange

HySOx is a fluorescent probe that shows excellent selectivity against hypochlorous acid (HOCl). Hypochlorous acid is one of the various reactive oxygen species (ROS) including hydroxyradical (・OH), superoxide (O2－・), hydrogen peroxide (H2O2), singlet oxygen (1O2), nitric oxide (・NO), and peroxynitrite (ONOO－). HySOx is almost non-fluorescent in neutral solutions. When HySOx reacts with hypochlorous acid, it generates a highly fluorescent product (with excitation maximum: 553 nm and emission maximum: 574 nm).

 

Available through Merck KGaA (Darmstadt, Germany) as:
SCT034 BioTracker™ 574 Red HOCL Dye

Products

Code No. Product Name Size Merck CAT No. Merck ( Millipore / Sigma Aldrich )
Product Name
GC3006-01 HySOx 20 μg × 5 SCT034 BioTracker 574 Red HOCL Dye

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  • Protocol

  • SDS

  • Product Information

    HySOx
    Product Information

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    Properties of HySOx

    Product Name Target Cell permeability Reacivity  Absmax (nm)  FLmax (nm)
    HySOx HClO Yes irreversible 553 574

     

    Selectivity of HySOx


    Fluorescence intensity of HySOx increases up to ~170-fold when it reacts with hypochlorous acid. Other ROS (superoxide, peroxynitrite, nitric oxide, singlet oxygen, and hydrogen peroxide) do not increase the fluorescent intensity of HySOx under identical conditions.

    Measurement conditions

    • Fluorescent intensities of 5 µM HySOx were measured after the addition of various ROS in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1% DMF as a cosolvent.
    • Fluorescent intensities were measured at 574 nm, with excitation at 553 nm, slit width 2.5 nm, photon multiplier voltage 700V, using HITACHI F-2700 Fluorescence Spectrophotometer.
    ROS generating conditions

    HOCl:  NaOCl 5 μM
    ・OH:  50 µM Fe(ClO4)2, H2O2 100 μM
    ONOO－:  HOONO 5 μM
    ・NO:  NOC18 5 μM
    O2-・:  KO2
    H2O2:  H2O2 100 μM
    PB:   phosphate buffer  (Negative control)

     

    Dose-dependent changes in the spectrum and intensity of HySOx fluorescence

    Figure 2. Fluorescent spectrum changes of 5 µM HySOx solution in various concentrations of hypochlorous acid.

    The reaction of HySOx with hypochlorous acid saturates at the molar ratio of 1:2.
    Inset, fluorescent intensities of HySOx against hypochlorous acid concentrations.

    Measurement conditions

    Fluorescent spectra of 5 µM HySOx were measured after the addition of hypochlorous acid (0-15 µM) in 0.1 M sodium phosphate buffer at pH 7.4 containing 0.1 % DMF as a cosolvent.

    Inset, fluorescent intensities of 5 µM HySOx measured at 574 nm with 0-5 µM of incremental hypochlorous acid concentrations. The solutions were excited at 553 nm with slit width 2.5mm, and measured with photon multiplier voltage of 700V, using HITACHI F-2700 Fluorescence Spectrophotometer.

     

  • Observation of hypochlorous acid production in U937 cells during phagocytosis

    HySOx
    Observation of hypochlorous acid production in U937 cells during phagocytosis

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    Observation of hypochlorous acid production in U937 cells during phagocytosis


    Production of hypochlorous acid during the phagocytosis.

    Procedure

    Induced differentiation of U937 cells by adding PMA.
    Dissolve HySOx with DMF to prepare 1 mM solution.
    DMF solution was diluted with HBSS to prepare 5 μM reaction solution.
    Removed medium from the cell culture dish, and rinsed cells with HBSS twice.
    Added the reaction solution to the cell culture dish and cultured for 30 minutes at 37oC 5% CO2.
    Removed the reaction solution and rinse cells with HBSS twice.
    Replaced with new HBSS, added Zymosan A and started time lapse imaging for 30 minutes.

FAQ

  • Q Tell me about the organic solvent to dissolve fluorescent probes.
    A

    Many fluorophores and activatable probes have low solubility to water. Therefore, to prepare a solution in millimolar-cocentrations, use organic solvent described in the protocol document in each products. For most products, dimethyl sulfoxide (DMSO) is a first choise. For some produicts, N,N-dimethylformamide (DMF) is recommended.

    For NHS products, anhydrous DMSO is recommended, because NHS is unstable in aqueous solutions. Use commercially available anhydrous DMSO, or prepare anhydrous DMSO by adding vacuum dried molecular sieves 3A to DMSO.

    Quality of organic solvents usually reduces upon moisture absorption, oxidation, or UV irradiation. Degradated organic solvents increase the fluorescence background or reduce the reactivity when use with some activatable probes. To avoid the degradation, it is recommended to dispense DMSO to small vials and store in a deep freezer and use up each of the vial after once the vial have opened. Avoid to store organic solvents in moist conditions, in high temperature, and under strong lights. 

  • 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

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