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Invitrogen™ Carboxy-H2DCFDA (general oxidative stress indicator)
Description
6-carboxy-2',7'-dichlorodihydrofluorescein diacetate is chemically reduced, acetylated form of fluorescein used as indicator for reactive oxygen species (ROS) in cells. This nonfluorescent molecule is readily converted to green-fluorescent form when acetate groups are removed by intracellular esterases and oxidation (by the activity of ROS) occurs within cell. Upon cleavage of the acetate and ester groups by intracellular esterases and oxidation, both analogs form carboxydichlorofluorescein, with additional negative charges that impede its leakage out of the cell.
- Ex/Em: approx. 492 - 495/517–527nm
- Product is air sensitive (store under dry argon or nitrogen)
- Product may be dissolved in DMSO, DMF, or ethanol for use
- Indicator is cell permeant
- Fluorescence can be monitored using a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein
- Carboxylated H2DCFDA analog (carboxy-H2DCFDA), which has two negative charges at physiological pH, and its di(acetoxymethyl ester) available separately
Refer to the Molecular Probes™ Handbook for additional product information.
Cell Analysis, Cell Metabolism, Cell Viability, Proliferation and Function, Nitro-Oxidative Stress, Reactive Oxygen Species
Order Info
Shipping Condition: Room Temperature
Specifications
Specifications
| Quantity | 25 mg |
| Product Type | ROS Indicator |
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.