Ethidium Bromide

Stain with ethidium bromide or another suitable DNA dye, identify the amplicon, and excise the band from the gel with a minimal amount of agarose.

From: Methods in Cell Biology , 2010

RECOMBINANT DNA CLONING

SUSAN J. KARCHER , in Molecular Biology, 1995

Ethidium Bromide Safety

Ethidium bromide is a valuable aid for visualizing DNA in gels, but it is considered a "powerful mutagen with moderate toxicity" ( MacGregor and Johnson, 1977). The greatest safety concern is during the preparation of a stock from the powdered ethidium bromide.

CAUTION: Use great care when weighing out powdered ethidium bromide. Ethidium bromide is a mutagen and probable carcinogen. Ethidium bromide is toxic. Wear gloves and a face mask when working with ethidium bromide in powdered form. Wipe the area with a damp cloth after the work with ethidium bromide powder is complete. A safer alternative is to purchase ethidium bromide in solution. This eliminates the hazards of working with the powdered form. Always wear gloves when working with ethidium bromide and ethidium-stained gels.

Also, while wearing gloves after handling ethidium bromide, be careful not to touch and thereby contaminate other surfaces. Be sure to wash hands with soap and warm water after working with ethidium bromide.

At present, the best way of disposing of ethidium bromide is not clear. There are several studies that suggest different methods to inactivate ethidium bromide. One study (Lunn and Sansone, 1987) compared eight methods of destroying ethidium bromide and found that treatment with sodium nitrite and hypophosphorus acid was most effective in destroying ethidium bromide. A common practice is to add sodium hypochlorite (bleach) to ethidium bromide solutions before disposing of them down the drain. The Lunn and Sansone (1977) study found that bleach treatment yields products that are quite mutagenic and recommends bleach not be used. On the other hand, Quillardet et al. (1988) report that bleach treatment reduces ethidium bromide to 20% of its initial mutagenicity or 0.1% of its initial mutagenicity if an activating mixture is not used in the mutagenicity test. Quillardet recommends using potassium permanganate/hydrochloric acid to destroy ethidium bromide. Bensaude (Quillardet et al., 1988) suggests adsorption of ethidium bromide to activated charcoal followed by incineration, because ethidium bromide decomposes at 262°C.

Some papers recommend that ethidium bromide be treated with KMnO4 to inactivate it. KMnO4 must be handled in a hood. Other studies suggest that the by-product of the KMnO4 treatment of ethidium bromide is even more mutagenic than ethidium bromide. Stained gels should be placed in plastic bags before disposal. Some schools have a solid waste program where ethidium bromide-stained gels are collected and incinerated.

Check with your local safety officer or local waste management control to determine how to dispose of ethidium bromide solutions and stained gels and about the disposal of other DNA stains such as methylene blue.

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Advances in Radiation Biology

Lawrence Grossman , in Advances in Radiation Biology, 1974

d Ethidium bromide-treated DNA

Ethidium bromide, which intercalates between base pairs in DNA, induces cytoplasmic respiratory "petite" mutations in yeast in the absence of growth ( Slonimiski et al., 1968). This effect is specific for this intercalating agent and results in a shortening of mitochondrial DNA (Goldring et al., 1970). Of the several nuclease activities identified in yeast mitochondria, only one is active on DNA duplexes previously treated with ethidium bromide and other intercalating dyes (Paoletti et al., 1972). It appears that one of the nuclease activities may be responsible for the yeast mitochondrial DNA degradation induced by ethidium bromide.

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Fluorescent Imaging of Nucleic Acids and Proteins in Gels

VICTORIA L. SINGER , RICHARD P. HAUGLAND , in Fluorescent and Luminescent Probes for Biological Activity (Second Edition), 1999

4.3.1 Ethidium bromide

Ethidium bromide is a phenanthridine monomer dye ( Fig. 4.1), which exhibits a 20- to 25-fold fluorescence enhancement upon binding to double-stranded DNA (LePecq & Paoletti, 1967). The dye intercalates between adjacent base pairs in the double-stranded DNA molecule (Reinhardt & Krugh, 1978). The fluorescence enhancement observed upon ultraviolet light excitation of intercalated ethidium bromide is also thought to be due in part to energy transfer from the bases of the DNA to the dye (LePecq & Paoletti, 1967). The primary sequence of the DNA has relatively little effect on nucleic acid binding, making this dye a good general-use stain. The dye penetrates electrophoretic gels rapidly and efficiently. Ethidium bromide can be precast in gels, used as a poststain, and is also occasionally used as an RNA prestain in formaldehyde-agarose gel electrophoresis.

Figure 4.1. Structure of ethidium bromide.

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General Perspective – The Future of Drug Discovery

L. Gutiérrez , ... J.M. de la Fuente , in Comprehensive Medicinal Chemistry III, 2017

1.09.4.2.4 DNA intercalating fluorescent agents

Ethidium bromide, propidium iodine (PI) and 7- amino-actinomycin D (7-AAD) are three red fluorescent dyes that intercalate into DNA of membrane-damaged cells ( Fig. 11 ). Ethidium bromide is considered carcinogenic, that is the reason why it is currently less used. PI and 7-AAD are both excited at 488   nm but the emission range(625   nm for PI and 655   nm for 7-AAD 655   nm), makes the difference, PI has significant spectral overlap with the fluorophore PE (Phycoerythrin) whereas 7-AAD no. Thus, 7-AAD is favorite in multiparameter staining experiments.

Fig. 11. Schematic representation of intercalating DNA agents staining nucleus of dead cells.

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Mechanisms of DNA Recombination and Genome Rearrangements: Intersection between Homologous Recombination, DNA Replication and DNA Repair

Rajula Elango , ... Anna Malkova , in Methods in Enzymology, 2018

3.4.2 Solutions and Reagents

•

Ethidium bromide (Sigma-Aldrich, #1239-45-8)

•

20× SSC pH 7: 175.3   g NaCl, 88.2   g sodium citrate, 900   mL MilliQ water

•

Alkaline solution: 20   g NaOH (RPI, #1310-73-2), 87.7   g NaCl (RPI, #7647-14-5) in 1   L of MilliQ water

•

Neutralization solution: 87.7   g NaCl (RPI, #7647-14-5), 121.4   g Tris base (RPI, #77-86-1) in 1   L of MilliQ water, pH 7

•

1× TBE: dilute 5   × TBE by adding 200   mL 5   × TBE in 1   L of MilliQ water

•

1% agarose gel: 1   g of agarose (Sigma-Aldrich, #A0169) in 100   mL of 1   × TBE

•

QIAGEN gel extraction kit: (QIAGEN, #28704)

•

P32-dCTP (Perkin Elmer, #BLU513H250)

•

RmT Random primer kit (Agilent, #300392)

•

Hybridization buffer: 50   mL of 0.5 M sodium phosphate buffer pH 7.2 (Fisher, #S374-1), 25   mL of 20% SDS (Fisher, #BP166-5), 200   μL of 0.5 M EDTA pH 8. Make up to 100   mL using MilliQ water

•

Low stringency wash buffer: 2   × SSC, 0.1% SDS

•

MilliQ water

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Transplantation of Myelin Forming Cells

V. Tepavčević , A. Baron-Van Evercooren , in Encyclopedia of Neuroscience, 2009

Focal demyelination

Lysolecithin and ethidium bromide . Focal demyelination areas can be induced in adult animals by injection of gliotoxic agents into predetermined white matter areas specifically killing glial cells but preserving the majority of the axons. The most commonly used toxins are lysophosphatidyl choline or lysolecithin (LPC) and ethidium bromide (EB). Lesions induced by LPC and EB show a limited amount of inflammation, usually associated with myelin debris removal, and have been used to answer basic questions regarding the remyelinating potential of different cell preparations. EB is a DNA intercalating agent which kills astrocytes, oligodendrocytes, and oligodendrocyte precursors. LPC is a membrane-solubilizing agent which has a particular toxicity for myelin but spares some oligodendrocytes and possibly some of their precursors. As a result, demyelination in EB lesions is more extensive and the remyelination process is slower than in the LPC model. Toxin-induced lesions do not result in behavioral deficits unless they are targeted to the cervical spinal cord. The advantage of these focal models is that the predetermined location of demyelinating lesions facilitates cell transplantation experiments. Cells can be placed either directly in a lesion to evaluate the remyelinating capacity of the preparation or at a distance from the lesion to examine migration. Xenograft rejection can be bypassed, inducing focal lesions in immunocompromised animals such as nude mice or rats.

Focal lesions induced in rodents undergo spontaneous remyelination. In order to create a situation in which endogenous remyelination is absent, William Blakemore's laboratory at Cambridge University developed a model in which local X-irradiation (40   Gy) of the spinal cord prior to or soon after EB injection is used to deplete the oligodendrocyte precursors present in the tissue, abolishing endogenous remyelination of the lesion for at least 6   weeks. Use of this nonrepairing lesion model overcomes the need to employ graft-specific markers to evaluate remyelination potential of the engrafted preparation since any remyelination observed can be attributed to transplanted cells ( Figures 2(c) and 2(d) ).

Focal lesions induced in rodents are small compared to demyelinating lesions in humans. In order to obtain a better understanding of human demyelinating conditions, nonhuman primate models of gliotoxin-induced demyelination have been developed in which lesions are targeted to a variety of CNS structures. These models reveal that LPC injection leads to chronic demyelination in the macaque optic nerve but acute demyelination followed by partial remyelination in the spinal cord ( Figure 3 ).

Figure 3. Electron microscopy images of lysophosphatidyl choline (LPC)-induced lesions in the macaque central nervous system. (a) Six weeks following injection of LPC into macaque spinal cord, newly formed myelin sheaths are evident adjacent to the astrocyte processes (As). (b) A bundle of demyelinating axons with only one axon remyelinated in the macaque optic nerve 6   weeks following LPC injection. Magnification: a, ×1300; b, ×14500. Figures kindly provided by Corinne Bachelin. Reproduced from Failure of remyelination in the nonhuman primate optic nerve, vol. 15, No.3, 2005, pp.198–207. Copyright (2005 and Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.); Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.

Cuprizone. Cuprizone intoxication of C57 black 6 mice results preferentially in demyelination of the corpus callosum. Although restricted to a specific structure in the brain, cuprizone lesions are far more widespread than the previously mentioned focal lesions. The advantage of this model is the possibility to achieve chronic demyelination by extending the treatment. Although appealing, this model has not been used for cell therapy.

Focal models of inflammatory demyelination. Several focal models of inflammatory demyelination are also available. Injections of antibodies into the oligodendocyte/myelin epitope such as galactocerebroside and myelin oligodendrocyte protein, in combination with complement have been used to induce focal demyelination and these are characterized by more extensive inflammation than the toxin models. Lipopolysaccharide injections into rat spinal cord also result in focal lesions of primary demyelination and infiltration of polymorphonuclear cells. Targeting of experimental autoimmune encephalomyelitis (EAE) lesions to the rat spinal cord or cortex has been achieved. Whereas spinal cord focal EAE lesions are characterized by extensive inflammation even at 1   month postinduction and behavioral deficits, cortical lesions show rapid remyelination. Except for the myelin-complement model, these models have seldom been used for transplantation studies.

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Laboratory Methods in Cell Biology

Shilo M. Smith , ... Yiqun G. Shellman , in Methods in Cell Biology, 2012

5.3 Step 3—Add EB/AO Dye Mix Working Solution to the Wells to be Analyzed

Overview Add EB/AO dye mix working solution and perform the assays.
Duration Less than 5 min for each well. Total duration will depend on the number of samples to be analyzed.
Procedure
3.1 Add 16 μL of EB/AO dye mix working solution into a well containing 150 μL media. Adjust the volume to be added proportionally if needed.
3.2 Wait for dye to diffuse and enter cells (about 1–2 min).
3.3 Examine the status of cells under the microscope, using the following criteria to determine whether the cells are live, apoptotic and necrotic:
A) Live cells have normal nuclei staining, which present green chromatin with organized structures.
B) Apoptotic cells contain condensed or fragmented chromatin, and they can be green or orange.
C) Necrotic cells have similar normal nuclei staining as live cells except the chromatin is orange instead of green.
3.4 If quantification is desired, count minimum of 100 total cells for each well to obtain the percentage of cells are live, apoptotic, or necrotic.
Tips 1. Different cell types and cell lines will have different morphology, and thus, it is very important to observe what the normal cells look alike before starting analyzing the treated cells. See Fig. 2 for example of the cell morphology for each category.
2. The EB/AO dye mix will eventually kill the cells, so only pipette a few wells at a time so that you can analyze them before they die. In addition, pipette slowly or you may wind up disturbing debris.

See Fig. 2 for example of the cell morphology for each category.

FIGURE 2. Examples of cell morphology for each category of cells. See the color plate.

Keywords

Keyword Class Keyword Rank Snippet
Methods
List the methods used to carry out this protocol (i.e. for each step).		 1. Apoptosis
2. Cell viability and cell death
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4
5
Process
List the biological process(es) addressed in this protocol.		 1. Apoptosis
2. Cell viability and cell death
3
4
5
Organisms
List the primary organism used in this protocol. List any other applicable organisms.		 1. Any animal tissue cultures in vitro
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3
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Pathways
List any signaling, regulatory, or metabolic pathways addressed in this protocol.		 1. Any pathway that affects cell viability, cell death, and apoptosis
2
3
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Molecule roles
List any cellular or molecular roles addressed in this protocol.		 1. Any molecule role that affects cell viability, cell death, and apoptosis
2
3
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Molecule functions
List any cellular or molecular functions or activities addressed in this protocol.		 1. Any function that affects cell viability, cell death, and apoptosis
2
3
4
5
Phenotype
List any developmental or functional phenotypes addressed in this protocol (organismal or cellular level).		 1. Any step that affects cell viability, cell death, and apoptosis
2
3
4
5
Anatomy
List any gross anatomical structures, cellular structures, organelles, or macromolecular complexes pertinent to this protocol.		 1
2
3
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5
Diseases
List any diseases or disease processes addressed in this protocol.		 1. Cancer and cancer treatment
2.Any diseases might be involved in alterations in viability, cell death, and apoptosis
3
4
5
Other
List any other miscellaneous keywords that describe this protocol.		 1
2
3
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5

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Laboratory Methods in Enzymology: DNA

Sara Lopez-Gomollon , Francisco Esteban Nicolas , in Methods in Enzymology, 2013

3 Materials

n-Butanol

Ethidium bromide

Sodium acetate (NaOAc)

Ethanol

GlycoBlue™ (Ambion)

Molecular weight marker (e.g., 10   bp DNA ladder, invitrogen)

Tris base

Boric acid (H3BO3)

EDTA, disodium

Bromophenol blue

Xylene cyanol

Formamide

Ammonium acetate (NH4OAc)

Magnesium acetate (MgOAc)

40% acrylamide:bisacrylamide (19:1)

Urea

Ammonium persulfate (APS)

N,N,N′,N′-tetramethylethylenediamine (TEMED)

Ultrapure water (e.g., purified through a Milli-Q system)

Caution N-Butanol is irritating to the mucous membranes, upper respiratory tract, skin and especially the eyes. Avoid breathing the vapors. Wear appropriate gloves and safety glasses. Use in a chemical fume hood. Keep away from heat, sparks, and open flames.
Ethidium bromide is a powerful mutagen. Consult label for specific handling and disposal procedures. Avoid breathing the dust. Wear appropriate gloves when working with solutions that contain this molecule.
Formamide is teratogenic. The vapor is irritating to the eyes, skin, mucous membranes, and the upper respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood when working with concentrated solutions of formamide. Keep working solutions covered as much as possible.
Unpolymerized acrylamide is a potent neurotoxin and is absorbed through the skin. Polyacrylamide is considered to be nontoxic but it should be handled with care because it might contain small quantities of unpolymerized acrylamide.
APS and TEMED are extremely destructive to tissue of the mucous membranes and upper respiratory tract, eyes, and the skin. Wear appropriate gloves, glasses, and protective clothing. Always use in a chemical fume hood.

3.1 Solutions & buffers

Step 2. 5×   TBE

Component Final concentration Stock Amount
Tris base 890   mM 54   g
Boric acid 890   mM 27.5   g
EDTA, pH 8.0 20   mM 0.5   M 20   ml

Add water to 1   l

1×   TBE

Add 200   ml of 5×   TBE to 800   ml water

2×   Formamide loading buffer

Component Final concentration Amount
Formamide 95% 95   ml
H2O 5   ml
EDTA 5   mM 2.7   g
Bromophenol blue 0.1% 1   g
Xylene cyanol 0.1% 1   g

Ethidium bromide (10  mg   ml−   1)

Dissolve 10   mg of ethidium bromide in 1   ml water

Step 4.A. Elution buffer

Component Final concentration Amount
Ammonium acetate 0.5   M 3.85   g
Magnesium acetate 10   mM 0.14   g
H2O 100   ml

Step 4.B. 0.25×   TBE

Add 50   ml of 5×   TBE to 800   ml water

Step 5. 3M NaOAc, pH5.2

Dissolve 12.3   g of sodium acetate in 40   ml of water. Adjust pH to 5.2. Add water to a volume of 50   ml

75% ethanol

Mix 75   ml ethanol with 25   ml water

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Animal Models for the Study of Multiple Sclerosis

Robert H. Miller , ... Andrew V. Caprariello , in Animal Models for the Study of Human Disease, 2013

Ethidium Bromide

The toxic effects of ethidium bromide are due to its DNA intercalating properties; therefore, all nucleated cells are affected by this agent. The very first experiments that made use of this toxin demonstrated that a direct infusion of ethidium bromide into the cisterna magna resulted in a reproducible acute demyelinating lesion of the rat brain stem. 124–126 Over time, the mode of injection has changed, and ethidium bromide is now typically injected directly into white matter tracts as a saline solution that varies from 0.01% or 0.05–0.1% depending on the injection site. The lesions seen after ethidium bromide injections made in the dorsal spinal cord tend to be quite large compared to LPC-induced lesions and can involve almost the entire dorsal funiculus, extending up to 8 mm longitudinally. The caudal cerebellar peduncles of rats have also been targeted with this approach to address critical questions regarding the effects of age, sex, growth factors, and the role of microglia/macrophage activation on remyelination. 127–133

Although ethidium bromide injections are typically administered in either rats or mice, injection into the spinal cords of cats has also been shown to result in large demyelinating lesions. 134 The early response to ethidium bromide injection involves the destruction of astrocytes, oligodendrocytes, and OPCs while axons are typically spared. Soon after the initial generation of the lesions, a large number of macrophages are found in and around the lesioned area, and astrogliosis occurs only around the perimeter of the lesion leaving the center of the lesions essentially devoid of astrocytes. 134 After the clearance of myelin debris, remyelination is relatively rapid, although it occurs more slowly than after LPC-induced demyelination. In a comparative study in which either LPC or ethidium bromide-induced demyelinating lesions were examined in the caudal cerebellar peduncle of adult rats, extensive oligodendrocyte remyelination was already seen throughout LPC-induced lesions at 6 weeks postinjection, whereas a significant proportion of axons remained demyelinated in ethidium bromide-induced lesions. By 3.5 months postinjection, however, almost all axons had remyelinated in ethidium bromide lesions. 135 Interestingly, in contrast to LPC or anti-GalC-induced lesions where most remyelination is mediated by oligodendrocytes, a significant amount of remyelination in ethidium bromide-induced lesions is undertaken by Schwann cells. 135,136 Remyelinating Schwann cells are typically found in astrocyte-depleted areas of the demyelinated spinal cord, and it was initially assumed that they were derived from Schwann cells associated with peripheral nerves or spinal nerve roots in the vicinity. 137 A recent study, however, used in vivo fate mapping to demonstrate that after demyelinating injury, CNS-derived OPCs are able to generate Schwann cells, a surprising finding given that Schwann cells normally develop from the embryonic neural crest and are restricted to the peripheral nervous system. 138 One likely hypothesis is that in the absence of astrocyte signaling, cells specified for the oligodendrocyte lineage are diverted to become Schwann cells, suggesting that the commitment to a myelinating fate overrides the commitment to a specific cell type. The molecular mechanisms responsible for this regulation of cell fate are currently unclear.

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Animal Models for the Study of Multiple Sclerosis

Robert H. Miller , ... Andrew C. Caprariello , in Animal Models for the Study of Human Disease (Second Edition), 2017

7.1.2 Ethidium Bromide

The toxic effects of ethidium bromide are due to its DNA intercalating properties, therefore all nucleated cells are affected by this agent. The very first experiments that made use of this toxin demonstrated that a direct infusion of ethidium bromide into the cisterna magna resulted in a reproducible acute demyelinating lesion of the rat brain stem ( Reynolds and Wilkin, 1993; Yajima and Suzuki, 1979a, 1979b). Over time, the mode of injection has changed, and ethidium bromide is now typically injected directly into white matter tracts as a saline solution that varies from 0.01% or 0.05% to 0.1% depending on the injection site. The lesions following ethidium bromide injections in the dorsal spinal cord tend to be quite large compared to LPC-induced lesions and can involve almost the entire dorsal funiculus, extending up to 8mm longitudinally. The caudal cerebellar peduncles of rats have also been targeted with this approach in order to address critical questions regarding the effects of age, sex, growth factors, and the role of microglia/macrophage activation on remyelination (Penderis et al., 2003a; Li et al., 2005; Li et al., 2006; Zhang et al., 2012; Ibanez et al., 2004; Adamo et al., 2006; Penderis et al., 2003b; Sim et al., 2002).

Although ethidium bromide injections are typically made in either rats or mice, injection into the spinal cords of cats has also been shown to result in large demyelinating lesions (Blakemore, 1982). The early response to ethidium bromide injection involves the destruction of astrocytes, oligodendrocytes, and OPCs while axons are typically spared. Soon after, the initial generation of the lesions' large numbers of macrophages are found in and around the lesioned area and astrogliosis occurs only around the perimeter of the lesion leaving the center of the lesions essentially devoid of astrocytes (Blakemore, 1982). Following the clearance of myelin debris, remyelination is relatively rapid, although it occurs more slowly than after LPC-induced demyelination. In a comparative study where either LPC or ethidium bromide-induced demyelinating lesions were examined in the caudal cerebellar peduncle of adult rats, extensive oligodendrocyte remyelination was already seen throughout LPC-induced lesions at 6 weeks post injection whereas, a significant proportion of axons remained demyelinated in ethidium bromide-induced lesions. By 3.5 months postinjection, however, almost all axons had remyelinated in ethidium bromide lesions (Woodruff and Franklin, 1999). Interestingly, in contrast to LPC or anti-GalC-induced lesions where most remyelination is mediated by oligodendrocytes a significant amount of remyelination in ethidium bromide-induced lesions are undertaken by Schwann cells (Woodruff and Franklin, 1999; Blakemore, 2005). Remyelinating Schwann cells are typically found in astrocyte-depleted areas of the demyelinated spinal cord and it was initially assumed that they were derived from Schwann cells associated with peripheral nerves or spinal nerve roots in the vicinity (Franklin and Blakemore, 1993). A recent study, however, used in vivo fate mapping to demonstrate that after demyelinating injury, CNS-derived OPCs are able to generate Schwann cells, a surprising finding given that Schwann cells normally develop from the embryonic neural crest and are restricted to the peripheral nervous system (Zawadzka et al., 2010). One likely hypothesis is that in the absence of astrocyte signaling, cells specified for the oligodendrocyte lineage are diverted to become Schwann cells suggesting the commitment to a myelinating fate over rides the commitment to a specific cell type. The molecular mechanisms responsible for this regulation of cell fate are currently unclear. In MS, when remyelination occurs, it is largely mediated by oligodendrocytes and not Schwann cells thus the significance of the ethidium bromide model for therapeutic development is somewhat limited.

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