DCFH-DA (2,7-dichlorofuorescin diacetate) is a fluorescent probe without fluorescence that can freely cross the membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to form DCFH (dichlorofluorescin). In the presence of reactive oxygen species (ROS), DCFH is oxidized to DCF (dichlorofluorescein) which is a strong green fluorescent substance that cannot penetrate the cell membrane. DCF has a maximum wave peak near the excitation wavelength of 502 nm and the emission wavelength of 525 nm, and the intensity is proportional to the level of intracellular reactive oxygen species.
Reactive oxygen species (ROS) refers to the general term of substances that are composed of oxygen in the body or in the natural environment and contain oxygen and are active in nature: there is mainly a kind of excited oxygen molecule, namely singlet oxygen molecule or singlet oxygen molecule. Three oxygen-containing free radicals, namely superoxide anion radicals, hydroxyl radicals, and hydroperoxyradicals, two peroxides, namely hydrogen peroxide and lipid peroxide, and a nitrogen-containing oxide. Dihydroethidium can cross the cell membrane and enter the cell to bind to DNA or RNA, forming hydroethidium to produce red fluorescence. Dihydroethidium is mainly oxidized by superoxide anion type reactive oxygen species in cells. Dihydroethidium itself is blue fluorescence with a maximum excitation wavelength of 370 nm and a maximum emission wavelength of 420 nm. After dehydrogenation, dihydroethidium binds to RNA or DNA to produce red fluorescence with an excitation wavelength of 300 nm or 518 nm and an emission wavelength of 610 nm.
Reduced glutathione (GSH) can react with dithionitrobenzoic acid (DTNB) to produce thio-nitrobenzoic acid and glutathione disulfide. Nitromercaptobenzoic acid is a yellow compound which has the maximum absorption peak at 420 nm. The GSH content can be calculated by measuring the OD value at 420 nm.
Reduced GSH can react with Dinitrobenzoic acid (DNTB) to form a yellow complex which can be detected by colorimetric assay at 405 nm and calculate the reduced GSH content indirectly.
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