Affordably priced reagents for DNA detection by fluorescence.
7-Amino actinomycin D (7-AAD) is a non-permeant dye that can be used to identify non-viable cells. Cells with damaged plasma membranes or with impaired/no cell metabolism are unable to prevent the dye from entering the cell. Once inside the cell, the dyes bind to intracellular DNA producing highly fluorescent adducts which identify the cells as non-viable. It is widely used in flow cytometry. 7-AAD is excited by the 488 nm laser line of an argon laser with fluorescence detected above 650 nm. Although the emission intensity of 7-AAD is lower than that of PI, the longer wavelength emission may make it more useful for multiplexing assays in combination with other 488 nm-excited fluorochromes such as FITC and PE.
Acridine orange is a nucleic acid selective fluorescent cationic dye useful for cell cycle determination. It is cell-permeable, and interacts with DNA and RNA by intercalation or electrostatic attractions. When bound to DNA, it is very similar spectrally to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). The dye is often used in epifluorescence microscopy.
Actinomycin is a polypeptide antibiotic isolated from soil bacteria of the genus Streptomyces. It binds to DNA duplexes, thereby interfering with the action of enzymes engaged in replication and transcription. Actinomycin D is one of the old chemotherapy drugs which have been used in therapy for many years.
DAPI is a fluorescent stain that binds strongly to DNA. It is used extensively in fluorescence microscopy. Since DAPI passes through an intact cell membrane, it can be used to stain live cells besides fixed cells. For fluorescence microscopy, DAPI is excited with ultraviolet light. When bound to double-stranded DNA its absorption maximum is at 358 nm and its emission maximum is at 461 nm. One drawback of DAPI is that its emission is fairly broad. DAPI also binds to RNA although it is not as strongly fluorescent as it binds to DNA. Its emission shifts to around 400 nm when bound to RNA. DAPIs blue emission is convenient for multiplexing assays since there is very little fluorescence overlap between DAPI and green-fluorescent molecules like fluorescein and green fluorescent protein (GFP), or red-fluorescent stains like Texas Red. Besides labeling cell nuclei, DAPI is also used for the detection of mycoplasma or virus DNA in cell cultures.
Ethidium bromide is an intercalating agent commonly used as a nucleic acid stain in molecular biology laboratories for techniques such as agarose gel electrophoresis. It has almost 20-fold fluorescence enhancement upon binding to DNA. Single-stranded RNA can also be detected, since it usually folds back onto itself and thus provides local base pairing for the dye to intercalate. Ethidium bromide is a very strong mutagen, and may possibly be a carcinogen or teratogen.
The Hoechst stains are a family of fluorescent stains for labeling DNA in fluorescence microscopy. Because these fluorescent stains label DNA, they are also commonly used to visualize nuclei and mitochondria. Two of these closely related bis-benzimides are commonly used: Hoechst 33258 and Hoechst 33342. Both dyes are excited by ultraviolet light at around 350 nm, and both emit blue/cyan fluorescence light around an emission maximum at 461 nm. The Hoechst stains may be used on live or fixed cells, and are often used as a substitute for another nucleic acid stain, DAPI. The key difference between them is that the additional ethyl group of Hoechst 33342 renders it more lipophilic, and thus more able to cross intact cell membranes. In some applications, Hoechst 33258 is significantly less permeant. These dyes can also be used to detect the contents of a sample DNA by plotting a standard emission-to-content curve.
The long-wavelength tracer nuclear yellow is often combined with the popular retrograde tracer true blue for two-color neuronal mapping. In neuronal cells, nuclear yellow primarily stains the nucleus with yellow fluorescence, whereas true blue is a UV light–excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence. Both nuclear yellow and true blue are stable when subjected to immunohistochemical processing and can be used to photoconvert DAB into an insoluble, electron-dense reaction product. Bisbenzimide and Nuclear Yellow, when transported retrogradely through axons to their parent cell bodies, may migrate out of the axons and the cell bodies, as indicated by fluorescence of adjoining glial nuclei. This migration was found to take place both in vivo and in vitro during storage of the sections in water. When using the tracers in 1% concentrations the in vivo migration may be controlled by restricting the survival time. The in vitro migration may be prevented by rapid histological processing of the material.
Propidium iodide (PI) belongs to the same chemical class of ethidium bromide. As in the case of ethidium bromide its fluorescence is enhanced for 20-30-fold upon binding to nucleic acids. The fluorescence excitation maximum is red-shifted for 30–40 nm and the fluorescence emission maximum blue-shifted for 15 nm or so. PI also binds to RNA as DAPI and acridine orange. PI is commonly used for identifying dead cells in a population of cells and as a counterstain in multicolor fluorescent techniques. It can also be used to differentiate necrotic, apoptotic and normal cells. It is suitable for fluorescence microscopy, flow cytometry and fluorometry.
Thiazole orange is a fluorescent DNA-binding dye for reticulocyte analysis. Also useful for Plasmodium species analysis.
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