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Invitrogen™ CM-H2DCFDA (General Oxidative Stress Indicator)

Catalog No. C6827
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Catalog No. C6827 Supplier Invitrogen™ Supplier No. C6827
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Description

General oxidative stress indicator

CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells. This indicator exhibits much better retention in live cells than H2DCFDA. CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols. Subsequent oxidation yields a fluorescent adduct that is trapped inside the cell, thus facilitating long-term studies.

ROS Indicator Specifications:

  • Excitation: ∼492–495 nm
  • Emission: 517–527 nm
  • Product is air sensitive and should be stored under dry argon or nitrogen
  • Product may be dissolved in DMSO, DMF, or ethanol for use
  • Indicator is cell permeant (cell loading protocols are available in the literature)
  • Fluorescence can be monitored using a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein

    Find more ROS indicators

    We offer an assortment of Molecular Probes™ products for the generation of reactive oxygen species (ROS), including singlet oxygen, superoxide, hydroxyl radical and various peroxide and hydroperoxides, as well as for their fluorometric detection in solution. Review Generating and Detecting Reactive Oxygen Species—Section 18.2 in the Molecular Probes™ Handbook for more information on these products.

    Dissolve the dye using high-quality, anhydrous dimethylsulfoxide (DMSO), dimethylformamide (DMF), or 100% ethanol to prepare stock concentration up to 5 mM. Working solutions should be freshly prepared. This probe oxidizes readily in solution thus presence of moisture will facilitate the decomposition of the dye.

    • Order Info

    Shipping Condition: Room Temperature

    Specifications

    Quantity 20 x 50 μg
    Product Type ROS Indicator

    Frequently Asked Questions (FAQs)

    I want to assay cells for reactive oxygen species using carboxy-H2DCFDA, but I want to do so with a plate reader instead of microscope. Will it work?

    It has been done. The problem is that plate readers are less sensitive than microscopes, with far less signal-to-background difference. It is worth trying, but first optimize concentrations and loading times with control cells, use a plate with little to no autofluorescence, and possibly optimize the gain setting in order to get the best signal possible. But don't expect the same sensitivity, even with optimization.
    I have GFP-transfected cells and need to label for reactive oxygen species. Can I use H2DCFDA?

    This is not recommended as the two dyes overlap in the emission wavelength. There are other ROS reagents available in different wavelengths, such as CellROX Deep Red, which emits in the far-red range (665 nm), or dihydroethidium, which is emits in the visible red range (620 nm).
    I labeled my cell with CM-H2DCFDA for reactive oxygen detection, but upon illuminating the cell there is a significant increase in fluorescence in the control cells. Why?

    If the cell is overloaded with dye, the high intracellular concentration of the dye may lead to dye-dye quenching. Upon illumination, photobleaching will occur, which will reduce the dye-dye quenching and actually increase the fluorescence (for a while, but then it will start decreasing). To solve the problem, reduce the concentration and incubation time, and try a range of incubation times and concentrations.
    I need a formaldehyde-fixable reactive oxygen species detection assay. Is H2 DCFDA fixable?

    H2DCFDA and similar derivatives are not fixable. The same goes for dihydroethidium and dihydrorhodamine. However, CellROX Deep Red and CellROX Green are retained for a limited time upon fixation with formaldehyde. CellROX Green may be retained upon subsequent Triton X-100 permeabilization. Avoid the use of any acetone or alcohol-based fixatives or fixatives that include alcohol, such as formalin.

    Why don't I see a significant change in signal for my live-cell fluorescent indicator dye?

    Regardless of the type of live-cell indicator dye (e.g., calcium indicators, pH indicator, metal ion indicators), make sure there is no serum during the loading step, which can prematurely cleave dyes with AM esters and bind dyes non-specifically. Always optimize the dye concentration and staining time with a positive control before you run your test samples, to give the best signal-to-background. Always run a positive control with a buffer containing free ions of known concentration and an ionophore to open pores to those ions (for instance, for calcium indicators like Fluo-4 AM, this would include a buffer with added calcium combined with calcimycin, or for pH indicators, buffers of different pHs combined with nigericin). Reactive oxygen indicators, such as CellROX Green or H2DCFDA would require a cellular reactive oxygen species (ROS) stimulant as a positive control, such as menadione. Finally, make sure your imaging system has a sensitive detector. Plate readers, for instance, have much lower detector efficiency over background, compared to microscopy or flow cytometry.

    For Research Use Only. Not for use in diagnostic procedures.