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Invitrogen™ Carboxy-H2DCFDA (general oxidative stress indicator)
Description
General oxidative stress indicator
6-carboxy-2',7'-dichlorodihydrofluorescein diacetate is a chemically reduced, acetylated form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells. This nonfluorescent molecule is readily converted to a green-fluorescent form when the acetate groups are removed by intracellular esterases and oxidation (by the activity of ROS) occurs within the cell. We offer the carboxylated H2DCFDA analog (carboxy-H2DCFDA, Cat. No. C400), which has two negative charges at physiological pH, and its di(acetoxymethyl ester) (Cat. No. C2938). Upon cleavage of the acetate and ester groups by intracellular esterases and oxidation, both analogs form carboxydichlorofluorescein (Cat. No. C368), with additional negative charges that impede its leakage out of the cell.
ROS Indicator Specifications:
- Ex/Em: ∼492–495/517–527 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.
For Research Use. Not for human or animal therapeutic or diagnostic use.
Order Info
Shipping Condition: Room Temperature
Specifications
Specifications
| Quantity | 25 mg |
| For Use With (Application) | ROS |
| Detection Method | Fluorescence |
| Product Type | ROS Indicator |
| Excitation Wavelength Range | 492 to 495 nm |
| Emission Wavelength Range | 517 to 527 nm |
Frequently Asked Questions (FAQs)
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.
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).
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.
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.
For Research Use Only. Not for use in diagnostic procedures.