Description
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The kit is designed for reactive oxygen species (ROS) detection in cell cultures by flow cytometer, microscopy or fluorimeter
Background: Reactive Oxygen Species can be induced by some stress conditions like exposure to oxidant or drugs. This fact leads to oxidative stress. ROS induce damage in DNA, protein and lipids, with important consequences in cells. Cell permeant reagent 2’-7’dichlorofluorescin diacetate (DCFH-DA) is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity. DCFH-DA probe. After cell uptake, DCFH-DA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS 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. Cell permeant reagent Dihydroethidium (DHE) is a fluorogenic dye that is useful for the detection of reactive oxygen species (ROS). DHE has been shown to be oxidized by superoxide to form 2-hydroxyethidium (2-OH-E+) (ex 500-530 nm/em 590-620 nm) or by non-specific oxidation by other sources of reactive oxygen species (ROS) to form ethidium (E+) (ex 480 nm/em 576 nm). Cell permeant reagent Dihydrorhodamine 123 (DHR 123) is a fluorogenic dye that is useful for the detection of reactive oxygen species (ROS) such as peroxide and peroxynitrite. After cell uptake, DHR 123 is oxidized by ROS into a fluorescent compound. It seems that neither the superoxide, the NO, nor the hydrogen peroxide by themselves, are capable of oxidizing DHR. These ROS, are thought to combine with other cellular components such as cytochrome c oxidase or Fe2+ in order to oxidize DHR 123 to its fluorescent derivative Rhodamine 123. Rhodamine 123 can be detected by fluorimeter, flow cytometer or fluorescence microscope with a maximum excitation and emission spectra of 500 and 536 nm, respectively. It can be also detected by absorbance spectroscopy at 500 nm (ε = 78,800 M-1cm-1)..
