ASTM E2186-02a(2010)
(Guide)Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
Standard Guide for Determining DNA Single-Strand Damage in Eukaryotic Cells Using the Comet Assay
SIGNIFICANCE AND USE
A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks may be introduced in many ways, directly by genotoxic compounds, through the induction of apoptosis or necrosis, secondarily through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes (20-22). In addition to a linkage with cancer, studies have demonstrated that increases in cellular DNA damage precede or correspond with reduced growth, abnormal development, and reduced survival of adults, embryos, and larvae (16, 23, 24).
The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay (10 000), the assay can be performed on practically any eukaryotic cell type, and it has been shown in comparative studies to be a very sensitive method for detecting DNA damage (2, 27).
These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained (28). Only fluorescent staining methods will be described in this guide. The microscopic determination of DNA migrati...
SCOPE
1.1 This guide covers the recommended criteria for performing a single-cell gel electrophoresis assay (SCG) or Comet assay for the measurement of DNA single-strand breaks in eukaryotic cells. The Comet assay is a very sensitive method for detecting strand breaks in the DNA of individual cells. The majority of studies utilizing the Comet assay have focused on medical applications and have therefore examined DNA damage in mammalian cells in vitro and in vivo (1-4). There is increasing interest in applying this assay to DNA damage in freshwater and marine organisms to explore the environmental implications of DNA damage.
1.1.1 The Comet assay has been used to screen the genotoxicity of a variety of compounds on cells in vitro and in vivo (5-7), as well as to evaluate the dose-dependent anti-oxidant (protective) properties of various compounds (3, 8-11). Using this method, significantly elevated levels of DNA damage have been reported in cells collected from organisms at polluted sites compared to reference sites (12-15). Studies have also found that increases in cellular DNA damage correspond with higher order effects such as decreased growth, survival, and development, and correlate with significant increases in contaminant body burdens (13, 16).
1.2 This guide presents protocols that facilitate the expression of DNA alkaline labile single-strand breaks and the determination of their abundance relative to control or reference cells. The guide is a general one meant to familiarize lab personnel with the basic requirements and considerations necessary to perform the Comet assay. It does not contain procedures for available variants of this assay, which allow the determination of non-alkaline labile single-strand breaks or double-stranded DNA strand breaks (8), distinction between different cell types (13), identification of cells undergoing apoptosis (programmed cell death, (1, 17)), measurement of cellular DNA repair ra...
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Designation: E2186 − 02a(Reapproved 2010)
Standard Guide for
Determining DNA Single-Strand Damage in Eukaryotic Cells
Using the Comet Assay
This standard is issued under the fixed designation E2186; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope cellular DNA repair rates (10), detection of the presence of
photoactive DNA damaging compounds (14), or detection of
1.1 This guide covers the recommended criteria for per-
specific DNA lesions (3, 18).
formingasingle-cellgelelectrophoresisassay(SCG)orComet
1.3 This standard does not purport to address all of the
assay for the measurement of DNA single-strand breaks in
safety concerns, if any, associated with its use. It is the
eukaryotic cells. The Comet assay is a very sensitive method
responsibility of the user of this standard to establish appro-
for detecting strand breaks in the DNAof individual cells.The
priate safety and health practices and determine the applica-
majority of studies utilizing the Comet assay have focused on
bility of regulatory requirements prior to use.
medical applications and have therefore examined DNAdam-
1.4 This guide is arranged as follows:
age in mammalian cells in vitro and in vivo (1-4). There is
increasing interest in applying this assay to DNA damage in
Section
freshwater and marine organisms to explore the environmental
Scope 1
implications of DNA damage.
Referenced Documents 2
1.1.1 The Comet assay has been used to screen the geno-
Terminology 3
Summary of Guide 4
toxicityofavarietyofcompoundsoncellsinvitroandinvivo
Significance and Use 5
(5-7), as well as to evaluate the dose-dependent anti-oxidant
Equipment and Reagents 6
(protective) properties of various compounds (3, 8-11). Using
Assay Procedures 7
Treatment of Data 8
thismethod,significantlyelevatedlevelsofDNAdamagehave
Reporting Data 9
been reported in cells collected from organisms at polluted
Keywords 10
sites compared to reference sites (12-15). Studies have also Annex A1
References
found that increases in cellular DNAdamage correspond with
higher order effects such as decreased growth, survival, and
2. Referenced Documents
development, and correlate with significant increases in con-
2.1 ASTM Standards:
taminant body burdens (13, 16).
E1706TestMethodforMeasuringtheToxicityofSediment-
1.2 This guide presents protocols that facilitate the expres-
Associated Contaminants with Freshwater Invertebrates
sion of DNA alkaline labile single-strand breaks and the
E1847Practice for Statistical Analysis of Toxicity Tests
determination of their abundance relative to control or refer-
Conducted Under ASTM Guidelines
ence cells. The guide is a general one meant to familiarize lab
personnel with the basic requirements and considerations
3. Terminology
necessary to perform the Comet assay. It does not contain
3.1 Thewords“must,”“should,”“may,”“can,”and“might”
proceduresforavailablevariantsofthisassay,whichallowthe
have very specific meanings in this guide. “Must” is used to
determination of non-alkaline labile single-strand breaks or
expressthestrongestpossiblerecommendation,justshortofan
double-stranded DNA strand breaks (8), distinction between
absolute requirement. “Must” is only used in connection with
different cell types (13), identification of cells undergoing
factors that relate directly to the acceptability of the test.
apoptosis (programmed cell death, (1, 17)), measurement of
“Should” is used to state that the specific condition is recom-
mended and ought to be met if possible.Although violation of
on “should” is rarely a serious matter, the violation of several
ThisguideisunderthejurisdictionofASTMCommitteeE50onEnvironmental
will often render the results questionable. Terms such as “is
Assessment,RiskManagementandCorrectiveActionandisthedirectresponsibility
of Subcommittee E50.47 on Biological Effects and Environmental Fate.
Current edition approved March 1, 2010. Published May 2010. Originally
approved in 2002. Last previous edition approved 2002 as E2186–02A. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/E2186-02AR10. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2186 − 02a (2010)
desirable,” “is often desirable,” and “might be desirable” are 3.2.12 single-stranded DNA, n—linear polymers of DNA
used in connection with less important factors. “May” is used resulting from the breaking of hydrogen bonds between
to mean “is (are) allowed to,” “can” is used to mean “is (are) complementary base pairs in double-stranded DNA.
able to,” and “might” is used to mean “could possibly.” Thus
3.3 Definitions of Terms Specific to This Standard:
the classic distinction between “may” and “can” is preserved
3.3.1 comet, n—namebasedontheappearanceofindividual
and “might” is never used as a synonym for either “may” or
stained nuclear DNA and associated relaxed or fragmented
“can.”
DNAmigrating out from the nuclear DNAobserved under the
3.2 Definitions: microscope following these assay procedures.
3.2.1 CCD camera, n—charge coupled device (CCD) cam-
3.3.2 DNAmigration distance, tail length, comet tail length,
era is a light sensitive silicon solid state device composed of
n—distance in microns between the leading edge of electro-
manysmallpixels.Thelightfallingonapixelisconvertedinto
phoretically migrating DNA and the closest edge of the
a charge pulse which is then measured by the CCD electronics
associated nuclear DNA (head).
and represented by a number.Adigital image is the collection
3.3.3 head, comet head, n—portionofacometcomprisedof
of such light intensity numbers for all of the pixels from the
the intact/immobile nuclear DNA.
CCD. A computer can reconstruct the image by varying the
3.3.4 tail,comettail,n—portionofacometcomprisedofthe
light intensity for each spot on the computer monitor in the
DNA migrating away from the intact/immobile nuclear DNA.
proper order. Such digital images can be stored on disk,
transmitted over a computer network, and analyzed using 3.3.5 tail moment, n—a calculated value used to express the
distribution of DNA migrating from the comet head. Image
image processing techniques.
analysissoftwareappliesanalgorithmtothedigitizedimageof
3.2.2 cell lysis, n—the process of breaking open a cell by
stained DNA and associated migrating DNA tail, which in
disruption of the plasma membrane.
essence defines the limits of the comet, subtracts background,
3.2.3 DNA, n—acronym for deoxyribonucleic acid, the sub-
and determines the boundaries and staining intensity of the
stance that is the carrier of genetic information found in the
nucleusandcomettail.Thecalculatedproductofthepercentof
chromosomes of the nucleus of a cell.
DNAinthetailandthetaillengthisdefinedasthetailmoment.
3.2.4 DNA denaturation, n—refers to breaking hydrogen
4. Summary of Guide
bonds between base pairs in double-stranded nucleic acid
moleculestoproducetwosingle-strandedpolynucleotidepoly-
4.1 Cells collected from organisms under different levels or
mers.
typesofstressaredispersedandimmobilizedinagarosegelon
microscope slides. The slides are placed in a solution to lyse
3.2.5 DNA lesion, n—a portion of a DNA molecule which
and disperse cell components, leaving the cellular DNA
has been structurally changed.
immobilized in the agarose. The DNA is denatured for a
3.2.6 DNA supercoiling, n—the condition of DNA coiling
specified period of minutes by immersing the slides in an
up on itself because its helix has been bent, overwound, or
alkaline solution. Strand breaks in the denatured cellular DNA
underwound.
results in higher degree of supercoil relaxation: the more
3.2.7 DNA supercoil relaxation, n—upon denaturation,
breaks, the greater the degree of relaxation. Given a sufficient
DNA strand breaks allow the supercoiled DNA to unwind or
degree of relaxation, the application of an electric field across
relax.
the slides creates a motive force by which the charged DNA
may migrate through the surrounding agarose, away from the
3.2.8 double-stranded DNA, n—a structural form of DNA
immobilized main bulk of cellular DNA. Following
where two polynucleotide molecular chains are wound around
electrophoresis, the alkaline conditions are neutralized by
each other, with the joining between the two strands via
rinsingtheslidesinaneutralpHbufferandfixationofslideand
hydrogen bonds between complementary bases.
its contents in ethanol. The DNA in the fixed slides is stained
3.2.9 electrophoresis, n—a method of separating large mol-
with fluorescent DNAstain and visualized using a fluorescent
ecules (such as DNAfragments or proteins) from a mixture of
microscope. Migration distance of DNA away from the
similar molecules. An electric current is passed through a
nucleus, comet tail length, can be measured by eye using an
medium containing the mixture, and each kind of molecule
ocular micrometer. Comet tail length, percent DNAin tail, tail
travelsthroughthemediumatadifferentrate,dependingonits
moment, and other DNA migration values can be calculated
electrical charge and size. Separation is based on these differ-
with the use of image analysis software.
ences. Agarose and acrylamide gels are the media commonly
used for electrophoresis of proteins and nucleic acids.
5. Significance and Use
3.2.10 eukaryotic cell, n—cell with a membrane-bound,
5.1 AcommonresultofcellularstressisanincreaseinDNA
structurally discrete nucleus and other well-developed subcel-
damage. DNA damage may be manifest in the form of base
lular compartments. Eukaryotes include all organisms except
alterations,adductformation,strandbreaks,andcrosslinkages
viruses, bacteria, and cyanobacteria (blue-green algae).
(19). Strand breaks may be introduced in many ways, directly
3.2.11 ocular micrometer, n—a graduated grid placed be- bygenotoxiccompounds,throughtheinductionofapoptosisor
tween the viewer’s eye and an object being observed under a necrosis, secondarily through the interaction with oxygen
microscope, to measure the object’s size. radicalsorotherreactiveintermediates,orasaconsequenceof
E2186 − 02a (2010)
excision repair enzymes (20-22). In addition to a linkage with cycle, cell turnover frequency, culture or growth conditions,
cancer, studies have demonstrated that increases in cellular and other factors that may influence levels of DNA strand
DNA damage precede or correspond with reduced growth, damage. Different cell types may have vastly different back-
abnormal development, and reduced survival of adults, ground levels of DNAsingle-strand breaks due to variations in
embryos, and larvae (16, 23, 24). excision repair activity, metabolic activity, anti-oxidant
concentrations, or other factors. It is recommended that cells
5.1.1 The Comet assay can be easily utilized for collecting
representingthosetobestudiedusingtheSCG/Cometassaybe
data on DNAstrand breakage (9, 25, 26). It is a simple, rapid,
examined under the light or fluorescent microscope using
and sensitive method that allows the comparison of DNA
stains capable of differentially staining different cell types.
stranddamageindifferentcellpopulations.Aspresentedinthis
Morphological differences, staining characteristics, and fre-
guide, the assay facilitates the detection of DNAsingle strand
quencies of the different cell types should be noted and
breaks and alkaline labile sites in individual cells, and can
compared to SCG/Comet damage profiles to identify any
determinetheirabundancerelativetocontrolorreferencecells
possiblecelltypespecificdifferences.Inmostcases,theuseof
(9, 16, 26). The assay offers a number of advantages; damage
homogenous cell populations reduces inter-cell variability of
to the DNA in individual cells is measured, only extremely
SCG/Comet values. The procedures for this assay, using cells
smallnumbersofcellsneedtobesampledtoperformtheassay
from many different species and cell types, have been pub-
(<10000), the assay can be performed on practically any
lishedpreviously (1, 2, 3, 5, 8, 10, 13, 14, 17, 18, 32-38).These
eukaryotic cell type, and it has been shown in comparative
references and others should be consulted to obtain details on
studies to be a very sensitive method for detecting DNA
the collection, handling, storage, and preparation of specific
damage (2, 27).
cell types.
5.1.2 These are general guidelines. There are numerous
procedural variants of this assay. The variation used is depen-
5.3 The experimental design should incorporate appropriate
dent upon the type of cells being examined, the types of DNA
controls, reference samples, and replicates to delineate the
damageofinterest,andtheimagingandanalysiscapabilitiesof
influence of the major sources of experimental variability.
thelabconductingtheassay.TovisualizetheDNA,itisstained
with a fluorescent dye, or for light microscope analysis the
6. Equipment and Reagents
DNA can be silver stained (28). Only fluorescent staining
6.1 Equipment:
methods will be described in this guide. The microscopic
6.1.1 Water Bath,setatatemperatureof35to40°Ctokeep
determination of DNA migration can be made either by eye
slidecoatingagaroseliquefiedduringthepreparationofslides.
using an ocular micrometer or with the use of image analysis
6.1.2 Centrifuge, capable of exerting a 600X g force and of
software. Scoring by eye can be performed using a calibrated
handling 1.5 mL microcentrifuge tubes. Lower g force will
ocular micrometer or by categorizing cells into four to five
require longer centrifugation times and refrigeration to mini-
classes based on the extent of migration (29, 30). Image
mize stress to cells.
analysis systems are comprised of a CCD camera attached to a
6.1.3 Electrophoresis Chamber and Power Supply, a sub-
fluorescent microscope and software and hardware designed
marine electrophoresis chamber, and a power supply able to
specifically to capture and analyze images of fluorescently
deliver a constant current up to 300 mAand a voltage gradient
stained nuclei. Using such a system, it is possible to measure
of 0.4 to 1.3 V/cm.
thefluorescenceintensityanddistributionofDNAinandaway
6.1.4 Fluorescent Microscope, DNA is visualized by stain-
from the nucleus (8). Using different procedural variants, the
ing with one of a number of fluorescent DNA stains su
...
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