Propidium bromide

The addition of ethidium bromide (EtBr) or propidium iodide (PI) allows the identification of DNA bands immediately after run on a transilluminator. Intercalation of the dyes into the double-stranded DNA yields fluorescence (EtBr excitation wavelength Aex 302 or 366 nm, emission wavelength Aem 590 nm PI Aex 530 nm, Aem 620 nm). 

Intercalators associate with dsDNA by insertion between the stacked base pairs of DNA [52], EtBr binds to dsDNA with little to no sequence specificity, with one dye molecule inserting for every 4-5 base pairs [53]. It also binds weakly via a non-intercalative binding mechanism only after the intercalative sites have been saturated [54], Propidium iodide (PRO) is structurally similar to ethidium bromide, and both dyes show a fluorescence enhancement of approximately 20-30 fold upon binding to dsDNA [41]. As well, their excitation maxima shift 30-40 nm upon binding due to the environment change associated with intercalation into the more rigid and hydrophobic interior of the double-stranded nucleic acid structure relative to aqueous solution [41]. 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

Metaphase chromosomes can be sorted by flow cytometry in the same way that cells can be sorted (see 10.7.5). Usually separation is based on the DNA content of ethidium bromide or propidium iodide stained chromosomes but Hoechst 33258 (which preferentially stains AT rich segments) and chromomycin A3 (which stains CG rich regions) can also be used (Davies et al., 1981). 

The diphenylamine method can be scaled down to increase sensitivity and fluorescent methods are available using diaminobenzoic acid, diamidino-phenylindole (DAP1), ethidium bromide or propidium iodide (Kissane and Robins, 1958 Hinegardner, 1971 Klotz and Zimm, 1972) which are sensitive down to 0.1 fig DNA. 

Other vital stains take advantage of different cellular properties which can be correlated with cellular physiology Propidium Iodide, Ethidium Bromide, Ethidium Monoazide, Calcofluor White have been widely used to indicate the presence of dead eukaryotes or prokaryotes cells. 2-(p-iodophenyl-)3)(p-nitro-phenyl)-5-phenyl tetrazolium chloride (INT) belongs to a class of stains which can be used to determine if a cell or hyphal compartments [180] can maintain an internal reducing environment (Fig. 20a). There are, however, still a large debate about the reliability of those techniques, depending upon the cells under consideration [181]. Calcofluor (Aex = 380 nm, Aem 420 nm) is a specific cell wall stain which enables to counts buds scars on Saccharomyces cerevisiae [29] to estimate the age of a cell. 

Add 8 g of cesium chloride to the 8 ml of supernatant and 0.6 ml of 2 mg/ml propidium iodide (PI). Some researchers have used ethi-dium bromide, but we recommend PI for maximum separation. Mix by inverting the tube, with Parafilm over top, until the CsCl has gone into solution. 

Vital dyes (trypan blue, eosin) fluorescence (Floechst, fluoresceins, rhodamine, ethidium bromide, propidium iodide) exogenous ( Cr release) endogenous (LDFI or alkaline phosphatase leakage, intracellular K+, lipid peroxidation)... 

Differential loss in cell membrane integrity between apoptotic and oncotic cells Combination assays Propidium iodide—YO-PRO-1 Acridine Orange-Ethidium Bromide Annexin V-Propidium iodide Useful for live, unfixed cells or unfixed organs only... 

Cyanine dyes and phenanthridine dyes, ethidium bromide and propidium iodide DAPl, Hoechst dyes , acridine orange, 7-AAD and hydroxystilbami-dine. 

SYBR dyes for sensitive detection in gels and blots, Chemically reactive SYBR dyes for bioconjugates. The three classes of classic nucleic acid stains are Intercalating dyes (ethidium bromide and propidium iodide), Minor-groove binders, (DAPI and the Hoechst dyes ). Miscellaneous nucleic acid stains with special properties (acridine orange, 7-AAD and hydroxystilbamidine). 39... 

Separation of mtDNA from nuclear DNA is achieved through gradients of CsCl mixed with a dye, generally ethidium bromide (EB), rarely 4 ,6-diamidino-2-phenylindole (DAPI) or propidium iodine (PDI). 

Baskic, D. Popovic, S. Ristic, P Arsenijevic, N. N. Analysis of cycloheximide-induced apoptosis in human leukocytes fluorescence microscopy using annexin V/propidium iodide versus acridine orange/ethidium bromide. Cell Biol. Int. 2006, 30, 924-932. 

Patel, D. J. Canuel, L. L. Biphasic helix-coil transition of the ethidium bromide.poly(dA-dT) and the propidium diiodide.poly(dA-dT) complexes ... 

Biggiogera, M. Biggiogera, F. F. Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level. J. Histochem. Cytochem. 1989,37,1161-1166. 

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