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Invitrogen™ H2DCFDA (H2-DCF, DCF)

Product Code. 11560326
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Product Code. 11560326 Supplier Invitrogen™ Supplier No. D399

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Description

Chemically reduced form of fluorescein used as an indicator for reactive oxygen species in cells

The cell-permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (also known as dichlorofluores cin diacetate) detects reactive oxygen species (ROS) in cells, for example to detect generation of reactive oxygen intermediates in neutrophils and macrophages. Upon cleavage of the acetate groups by intracellular esterases and oxidation, nonfluorescent H2DCFDA is converted to highly fluorescent 2',7'-dichlorofluorescein (DCF).

  • Ex/Em: ∽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

Refer to the Molecular Probes™ Handbook for additional product information.

Apoptosis, Cell Analysis, Cell Metabolism, Cell Viability, Proliferation and Function, Free Radical Detection, Nitro-Oxidative Stress, Reactive Oxygen Species

Order Info

Shipping Condition: Room Temperature

TRUSTED_SUSTAINABILITY

Specifications

Quantity 100 mg
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.

What is the recommended storage condition/shelf-life for H2DCFDA (H2-DCF, DCF) (Cat. No. D399)?

We guarantee stability of the product for a period of 3 months when stored as recommended in the freezer (-5 to -30 degrees C).
I am trying to label my cells with a reactive oxygen species (ROS) indicator dye, but I am not seeing a significant difference in signal. What could be happening?

First, make sure you have both a negative (untreated) and positive (ROS-induced) sample to compare. A good positive control can be the use of 100 µM menadione for one hour or 50 µM nefazodone for 24 hours. H2O2 can also be used, though it does not work well for CellROX dyes. Some dyes, such as H2DCFDA, require esterase cleavage, so don't incubate in the presence of serum (which contains esterases that can prematurely cleave the dye). If your positive control does not show significant change compared to the negative control, try increasing the concentration and label time for the dye. Our manuals give starting recommendations. Be sure to image your live cells as soon as possible. Only two dyes (CellROX Green and CellROX Deep Red) are retained with formaldehyde fixation. Finally, make sure you are using filters and instrument settings to match the excitation and emission spectra of the dye.

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

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