Fluorescent probes mix (DHE, DHR 123 and DCFH-DA) are a useful tool for measuring free radicals like Reactive Oxygen Species or Reactive Nitrogen Species. Is for that reason, that Multiprobe REDOX kit by BQCkit that contains the most used fluorescent probes, has a great advantage because you can measure different kinds of free radicals with just one product.
DCFH-DA probe is a fluorescent probe that penetrates in the cell. After cell uptake, cellular esterases deacetylates DCFH-DA to a non-fluorescent compound. This is due to the fact of ROS oxidizes this probe into 2’-7’dichlorofluorescein (DCF). DCF is a fluorescent compound which can be detected by fluorimeter, flow cytometer or fluorescence microscope with a maximum excitation and emission spectra of 495 nm and 529 nm respectively.
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Superoxide, oxidizes DHE probe (a non fluorescen probe ) to form 2-hydroxyethidium (2-OH-E+) (ex 500-530 nm/em 590-620 nm). Moreover, there are some other sources of reactive oxygen species (ROS) that can produce a non-specific oxidation in DHE to form ethidium (E+) (ex 480 nm/em 576 nm).
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DHR 123 probe
Free radicals can oxidize DHR 123 (a non fluorescent compound) to rhodamine. It seems that neither the superoxide, the NO, nor the hydrogen peroxide by themselves, are capable of oxidizing DHR. So, these ROS, are combined with other cellular components such as cytochrome c oxidase or Fe2+ in order to oxidize DHR 123 to its fluorescent derivative Rhodamine 123.
Fluorimetry, Flow cytometry and fluorescent microscopy, are useful techniques to measure Rhodamine 123 due to its fluorescence. This probe, has a maximum excitation and emission spectra of 500 and 536 nm, respectively.
Rhodamine 123 presents a slighltly colour, so absorbance spectroscopy is also a possible technique to detect this probe. The optimal wavelenght is 500 nm (ε = 78,800 M-1cm-1).
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