Methods 48 (2009) 46–53

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Methods
journal homepage: www.elsevier.com/locate/ymeth

Review Article

The comet assay: A sensitive method for detecting DNA damage in individual cells
Wenjuan Liao a, Michael A. McNutt b, Wei-Guo Zhu a,*
a
Department of Biochemistry and Molecular Biology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education),
Peking University Health Science Center, #38 Xueyuan Road, Beijing 100191, China
b
Department of Pathology, Peking University Health Science Center, #38 Xueyuan Road, Beijing 100191, China

a r t i c l e i n f o a b s t r a c t

Article history: The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells, and
Accepted 25 February 2009 the year 2009 represents the 25th anniversary of the first description of this methodology in 1984. Over
Available online 6 March 2009 time this method has been improved, but is still not completely standardized, and variations are cur-
rently in widespread use with emphasis on applications in research and genetic toxicology. Here we
Keywords: review the principles of the comet assay and cite key studies that have focused on this assay in the past
Comet assay 25 years. In addition, we present an example of how the comet assay was used in our laboratory for
Single cell gel
studying the induction of DNA damage in human lung cancer cells after differing doses of the cytosine
DNA strand breaks
DNA damage and repair
analog 5-aza-20 -deoxycytidine (5-aza-CdR). Finally, some insights into the potential of this assay in can-
cer research and work in related fields are offered.
Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction otic cells may be measured. It is based on quantification of the

Twenty-five years ago, two Swedish scientists – Ostling and
Johason – developed a new method of analyzing DNA damage for
use in biological research which imports the word ‘‘comet” from
the realm of astronomy as its name [1]. The comet assay is a meth-
od in which DNA lysis and electrophoresis are performed under
neutral conditions, and acridine orange is used for staining of the
DNA. This metaphore from astronomy is visually appropriate as
the resultant image obtained with this technique looks like a
‘‘comet” with a distinct head consisting of intact DNA, and a tail
which contains damaged or broken pieces of DNA. The amount of
DNA liberated from the head of the comet during electrophoresis
depends on the level of effect of the mutagen under evaluation.
Coincidently, the year 2009 has been designated the International
Year of Astronomy (IYA2009) by the United Nations to celebrate
the first use of the telescope by Galileo in astronomy – a momen-
tous event that initiated 400 years of astronomical discovery which
has profoundly effected our worldview. The utility of this assay
which bears the name of a celestial body which may have studied
by Galileo has proven to be so great, that we may perhaps expect
that it will be in use for a length of time equal to the scientific rev-
olution which was triggered by the telescope.
The comet assay, which is also referred to as the single cell gel
electrophoresis assay (SCG or SCGE assay), is a rapid and quantita-
tive technique by which visual evidence of DNA damage in eukary-
* Corresponding author. Fax: +86 10 82805079.
E-mail addresses: zhuweiguo@bjmu.edu.cn, zhuwei_guo@yahoo. com (W.-G.
Zhu).
denatured DNA fragments migrating out of the cell nucleus during
electrophoresis. This assay has gained widespread use in various
areas including human biomonitoring, genotoxicology, ecological
monitoring and as a tool for research into DNA damage or repair
in different cell types in response to a range of DNA-damaging
agents [2]. In this review we focus on how the comet assay has
been applied to the study of DNA damage and repair in human can-
cer cells in response to various DNA damage agents.
The human genome is constantly exposed to agents that dam-
age DNA. Three main categories of agents or mechanisms which in-
duce DNA damage have been classified by Hoeijmakers [3] as
follows: (1) environmental agents such as ultraviolet light (UV),
ionizing radiation and numerous genotoxic chemicals which cause
alterations in DNA structure; (2) products of normal cellular
metabolism which constitute an ongoing source of damage to
DNA integrity; and (3) certain chemical agents which bond to
DNA and tend to cause spontaneous disintegration of DNA under
physiological conditions. These represent diverse mechanisms of
DNA damage and give rise to a perplexing array of DNA lesions
which are harmful in general to the human genome and may lead
to development of cancer. Among such DNA damage, acute effects
arise from disturbed DNA metabolism and halt cell-cycle progres-
sion or cause cell death. Long term effects result from irreversible
DNA mutations. On the other hand, induction of DNA damage in
cancer cells is a well recognized therapeutic strategy for killing
cancer. However, drug resistance is a major obstacle in cancer che-
motherapy. After an initial therapeutic response, many patients
develop recurrent disease which is refractory to chemotherapy
due to the emergence of drug resistant cell clones. It is therefore

1046-2023/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2009.02.016
 W. Liao et al. / Methods 48 (2009) 46–53 47

essential to use an assay capable of detecting such subpopulations
of tumor cell. A unique property of the comet assay is its ability to
identify and quantify cellular heterogeneity in response to DNA-
damaging agents, and this assay was in fact the first assay devel-
oped which was capable of identifying DNA damage in single cells
[4]. For example, our laboratory has recently developed a model
therapeutic strategy for cancer treatment, in which the demethy-
lating agent 5-aza-20 -deoxycytidine (5-aza-CdR) acts in conjunc-
tion with the histone deacetylase inhibitor (HDACI) depsipeptide/
TSA to induce significant apoptotic cell death in human lung cancer
cell lines, and we used the comet assay to evaluate for the presence
and mechanism of DNA damage in this model [5–7].
The comet assay possesses a number of advantages as com-
pared to other genotoxicity tests. In addition to the capability of
this assay to identify DNA damage at the single cell level, other sig-
nificant advantages include its sensitivity for detecting low levels
of DNA damage, the requirement for only small numbers of cells
per sample, its ease of application and low cost, and the short time
needed to perform the assay. In addition this assay is flexible as it
can be used to evaluate for various types of DNA damage, and is
readily modifiable for adaptation to a variety of experimental
requirements [8]. It is therefore no wonder that the comet assay
has been used in many studies to investigate DNA damage and re-
pair in a wide range of tumor cells with a variety of DNA-damaging
agents.
2. Development and use of comet assay
When Ostling and Johason originally developed the comet assay
for evaluating DNA damage at the single cell level, their technique
consisted of lysing cells with detergents and high salt, and subject-
ing the liberated DNA to electrophoresis under neutral conditions.
DNA was then stained with the fluorescent dye acridine orange and
which gives a green emission quantifiable with a microscope pho-
tometer. The amount of the DNA liberated from the head of the co-
met depends on the effect of the mutagen which is used. However,
in this earliest version of the procedure, only double-strand breaks
(DSB) could be evaluated.
Singh et al. subsequently modified the protocol by inclusion of
unwinding of DNA under alkaline conditions (pH > 13). Cells were
lysed at pH 10 with 2.5 M NaCl, Triton X-100 and Sarkosyl for 1 h,
followed by treatment with alkali (0.3 M NaOH) and electrophore-
sis at high pH (>13). At this pH, increased DNA migration is associ-
ated with increased levels of single-strand breaks (SSB), SSB
associated with incomplete excision repair sites, and alkali-labile
sites (ALS) [9]. Because almost all genotoxic agents induce SSB
and/or ALS in numbers which are orders of magnitude greater than
DSB, this version of the assay offered greatly increased sensitivity
for identifying genotoxic agents. This method has been recom-
mended as the optimal version of the comet assay for use in iden-
tifying agents with genotoxic activity at the International
Workshop on Genotoxicity Test Procedures (IWGTP) [8].
Two years after development of the protocol by Singh et al., Ol-
ive introduced another alkaline version of this assay in which DNA
electrophoresis is carried out at a pH of 12.3, wherein cells were
lysed in weak alkali (0.03 M NaOH) for 1 h before electrophoresis
[10]. This variant of the assay was developed for optimal detection
of subpopulations of cells with varying sensitivity to drugs or radi-
ation. In fact, since the introduction of the alkaline (pH > 13) comet
assay in 1988, the breadth of application and the number of inves-
tigators using this technique have increased exponentially.
The most widely adopted modification of this technique has
been inclusion of enzymic digestion with specific DNA glycosy-
lase/endonuclease enzymes that identify broad classes of DNA
lesions [11]. In this manner it is possible to detect oxidized pyrim-
idines by digestion with endonuclease III (ENDOIII) and use
formamidopyrimidine-DNA glycosylase (FPG) to identify the major
purine oxidation product 8-oxouanine as well as other altered pur-
ines. The premutagenic lesion 7-hydro-8-oxo-20 -deoxyguanosine
(8-OHdG) is probably one of the most important lesions which
can be assayed with use of the FPG protein. There are also other en-
zymes which have been used less frequently in this manner,
including the 3-methyladenine DNA glycosylase II (Alk A) protein
which can be used for alkylation damage and T4 endonuclease V
for UV-induced lesions. Novel applications of the comet assay in-
clude identification of DNA–DNA crosslinks, and gene-specific
DNA damage with application of fluorescent in situ hybridization
methodology [12,13]. The comet assay can also be modified to
measure the DNA repair activity of cell extracts on gel-embedded
DNA substrate which harbor specific lesions. Collins and his
coworkers first designed this kind of experiment to measure the
capacity of human lymphocyte extract for site specific incision,
which is the initial step of DNA repair of oxidized bases [14]. The
comet assay has been applied in a broad range of scientific fields,
including genetic toxicology, ecotoxicology and DNA repair re-
search. Table 1 lists important historic events in the development

Table 1
Highlights of comet assay achievements and novel application in the past 25 years.

Year Development and achievement References

1984 First to develop a microgel electrophoresis technique (neutral method) for detecting DNA double-strands break at the level of the single cell [1]
1988 The single cell microgel electrophoresis under alkaline conditions (pH > 13) was first introduced for detecting DNA damage in single cells., which [9]
turned to be the most popular method used of comet assay
1990 Another alkaline version (pH = 12.3) was introduced [10]
1992 Provides a sensitive way to detect the short-lived intermediates in DNA repair in proliferating cells [17]
1993 The endonuclease III is used to detect oxidized pyrimidines [11]
1995 First time to described necrotic or apoptotic cells can result in comets with small or nonexistent head and large diffuse tails [26]
1996 The results of this study indicate that the comet assay is a useful tool for the detection of DNA–DNA cross-linking agents [34]
1997 T4 endonuclease V to recognize UV-induced cyclobutane pyrimidine dimers [35]
1997 First describe the method of combining the comet assay with FISH, and identify chromosome-specific areas on electrostretched DNA fibers and to [13]
determine their spatial distribution
1999 Developed a BrdUrd-comet assay which is simple and suitable for the accurate and automatable assessment of replicative integrity in very small [36]
numbers of mammalian cells, such as may be obtained by biopsy
2000 International guidelines published for application in genetic toxicology and biomonitoring [8]
2001 Measure DNA repair activity in cell extract. The incision efficiency of cell extracts from donors is measured on gel-embedded substrate DNA [14]
containing specific type of DNA lesions
2001 This report show that it was possible to show that apoptosis can generate typical comet pictures as soon as the cells enter the apoptosis process [37]
2002 An improved comet assay found endonuclease III, formamidopyrimidine-DNA glycosylase, and proteinase K additively enhance arsenic-induced [38]
DNA strand breaks in human cells
2003 It found that no nuclei were lost due to apoptotic nuclear fragmentation and DNA migration. Even the so-called ‘‘hedgehog” comet images with [29]
extreme DNA damage were not lost during liquid holding
2006 HOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII [39]
48 W. Liao et al. / Methods 48 (2009) 46–53
of the comet assay and breakthrough modifications over the
25 years it has been in use.
3. Experimental methods – general considerations
Several versions of the comet assay are currently in use. Because
the alkaline (pH > 13) version of this assay is both superior for eval-
uating a broad spectrum of DNA lesions, and maximizes sensitivity
for detection of low levels of damage, it has been chosen for
description in detail here as a useful general tool for monitoring
DNA damage. After a suspension of cells has been obtained, the ba-
sic steps in the assay include preparation of microscopic slides lay-
ered with cells embedded in an agarose gel, lysis of cells to liberate
the DNA, DNA unwinding, electrophoresis, neutralization of the al-
kali, DNA staining and scoring. Performance of these steps in a con-
sistent manner allows for reliable detection of DNA damage
induced by genotoxic agents. As a general rule, all buffers and re-
agents used in this assay should be prepared no more than
1 month in advance.
3.1. Slide preparation and cell lysis
The goal of slide preparation is to ensure easily visualized comets
with minimal background noise as well as to obtain uniform gels suf-
ficiently stable for subsequent manipulation. Generally, a ‘‘sand-
wich” gel where cells are contained in the middle layer of three
distinct layers of agarose is used (Fig. 1). Cells are suspended in
low-melting point (LMP) agarose (generally at 37 °C) in the middle
layer. In our laboratory, we have found that omitting the top (last)
layer of LMP agarose does not affect the final result, provided there
is complete subsequent neutralization of the alkali. An appropriate
sized coverslip is used to flatten out each molten agarose layer,
and the slides are often chilled during the process to enhance gelling
of the agarose. The concentration of cells in the agarose, as well as the
concentration of the agarose itself, are important parameters for
ensuring a successful analysis. Care must be taken with the number
of cells per visual field, as higher cell densities can result in a signif-
icant proportion of overlapping comets, especially at high rates of
DNA migration. On the other hand, higher agarose concentrations
can affect the extent of DNA migration.
After the gel containing cells has solidified, the slides are placed
in a lysis solution consisting of high salts and detergents generally
for at least 1 h. There are not many significant differences among
the various alkaline lysis methods, all of which employ high salt/
detergent lysis for a variable period (1–24 h). However, some cell
types may require the presence of a second detergent for complete
lysis, and this must be evaluated on a case by case basis. The lysis
solution is chilled prior to use, primarily to maintain the stability of
the agarose gel. Between lysis and alkali unwinding, the liberated
DNA can be incubated with proteinase K (PK) to remove residual
proteins or probed with DNA repair enzymes/antibodies to identify
specific classes of DNA damage (e.g. oxidative damage).
Fig. 1. A typical three-layer sandwich slide. Cells are suspended in low-melting
point (LMP) agarose and placed on a slide precoated with a layer of regular agarose.
After addition of the cell-containing layer, another layer of LMP agarose is added to
fill in any residual holes in the second layer and to increase the distance between
cells and the gel surface.
3.2. Alkali (pH > 13) unwinding
Prior to electrophoresis, the slides are incubated in alkaline
(pH > 13) electrophoresis buffer to produce single-stranded DNA
and to express ALS as SSB. The alkaline solution developed by Singh
et al. [9] consists of 1 mM EDTA and 300 mM sodium hydroxide,
pH > 13.0. This solution is still the one which is most frequently
used in comet studies, as it maximizes the expression of ALS as
SSB. Based on the protocol of Singh et al. (1988), an incubation per-
iod of 20 min is generally used. However, one method for testing
the adequacy of the alkali unwinding period is monitoring the ex-
tent of DNA migration in the control cells (identical to cells to be
tested, but with oxidative damage induced by a previously charac-
terized reagent such as H2O2) under different unwinding condi-
tions (length of time and buffer) and keeping all other steps
identical.
3.3. Electrophoresis
After alkali unwinding, the single-stranded DNA in the gel is
subjected to electrophoresis under alkaline conditions to produce
comets. The alkaline buffer used during electrophoresis is the same
pH > 13 buffer which is used during alkali unwinding. This tech-
nique differs from conventional DNA electrophoresis, since DNA
migration of only a fraction of the distance is required, and thus
only very short electrophoresis running time (5–30 min) and low
voltages (0.5–5.0 V/cm) are needed. The electrophoretic conditions
originally used in Singh’s protocol [9] were 25 V and 300 mA, with
DNA electrophoresis for 20 min. However, many variations in elec-
trophoretic conditions under use have subsequently been reported,
including a study from the original laboratory [15]. The optimal
voltage/amperage and duration of electrophoresis generally de-
pends on the extent of DNA migration seen in the control cells,
and range of migration under evaluation among the treated cells.
Electrophoresis has been conducted at temperatures ranging from
5 °C to room temperature, and the use of lower temperatures is
thought to provide increased reproducibility.
3.4. Neutralization
After electrophoresis, the alkali in the gels is neutralized by
rinsing slides with a suitable buffer. Three washes of trizma buffer
for 5 min each are typically used, however, when a high back-
ground is found during scoring, increased rinsing may be useful
in some situations. Under neutralization, the DNA strands sepa-
rated by alkaline treatment in the comet head will readily renature
due to their intact structure with supercoiled loops, while the DNA
in tail will remain single-stranded. After neutralization, slides can
be stained and comets scored. Alternatively the gel can be dried,
the slides stored, and the comets scored when convenient.
3.5. DNA staining and comet visualization
DNA is visualized by fluorescence microscopy after staining with
a DNA binding dye. Ethidium bromide (EB) [1,9] is probably the re-
agent most commonly used, followed by propidium iodide [16]
and 4,6-diamidino-2-phenylindole (DAPI) [17]. These dyes all bind
in different ways. EB which is a DNA intercalator, inserts itself into
the spaces between the base pairs of the double helix. DAPI is known
to form fluorescent complexes with double-stranded DNA, showing
a fluorescence specificity for AT, AU and IC clusters, and there is evi-
dence that DAPI binds to the minor groove of the DNA helix, and is
stabilized by hydrogen bonds between DAPI and acceptor groups
of AT, AU and IC base pairs. PI binds to DNA by intercalating between
the bases with little or no sequence preference at a stoichiometric ra-
tio of one dye per 4–5 base pairs of DNA. For some fluorescent dyes,
 W. Liao et al. / Methods 48 (2009) 46–53
antifade can be used to significantly reduce the rate of signal quench-
ing [18], allowing the same slide to be scored multiple times. In addi-
tion, there are a number of alternative stains including propidium
iodide, YOYO-1TM, SybrGold TM and TOTO TM, as well as non-fluo-
rescent techniques for visualizing comets based on staining with sil-
ver nitrate [19].
For microscopic evaluation, comet image magnification has gen-
erally varied from 160 to 600, and magnifications of 200 to
400 are most commonly used. The magnification which is most
appropriate depends on the type of cell under evaluation, the range
of distances of migration to be measured for given experimental con-
ditions, and the microscope and/or imaging system which is used.
3.6. Evaluation and interpretation of results
Several different attempts have been made to evaluate and
quantify comet formation patterns, and a variety of commercial
and freeware IA systems are available for assessing the resultant
images. Most commonly, the distance of DNA migration from the
body of the nuclear core is used to measure the extent of DNA
damage when dealing with relatively low damage levels. However,
this technique is not very useful in situations where DNA damage
is relatively high, as with increasing extent of DNA damage the tail
increases in fluorescent staining intensity but not in length. In
addition, a scoring method based on visual inspection was recom-
mended by Collins [2], and this method may be especially useful
for a laboratory which has no previous experience with the comet
assay due to its relative ease of use. There are five classes estab-
lished in that study ranging from 0 (no tail) to 4 (almost all DNA
in tail) and use of this system has been found to give quantitative
resolution which is sufficient for many purposes. Another popular
method for comet evaluation is referred to as ‘tail moment’. The
concept of tail moment (calculated as: measure of tail
length  measure of DNA in the tail) as a measure of DNA migra-
tion was introduced by Olive et al. [20]. This method incorporates
relative measurements of both the smallest detectable size of
migrating DNA (reflected by the length of the comet tail) and the
number of broken pieces of DNA (represented by the staining
intensity of DNA in the tail). In the end, the method chosen for
evaluation of DNA migration depends on the resources of the
investigator and the experimental design.
In selection of comets for evaluation, edges and areas around air
bubbles should be avoided, as these areas often display anoma-
lously high levels of damage. An additional point for caution is that
the comet assay has a limited dynamic range, and becomes satu-
rated when most of the DNA is in the tail. (Saturation is reached
at about the level of three breaks per 109 Da). This is not a problem
with the low levels of background damage found in normal cells.
However, when using lesion-specific endonucleases to measure
additional base damage, total damage scores may approach the
saturation limit, and the total enzyme-sensitive sites are likely to
be underestimated unless care is taken to calibrate the assay [21].
4. Use of the comet assay to evaluate DNA damage and repair in
lung cancer cells after differing doses of 5-aza-CdR
In order to illustrate the type of research situation in which the
comet assay may be of value, the following example is taken from
research which was done in our laboratory. This example serves to
underscore use of the comet assay to first determine whether DNA
damage has taken place under given experimental conditions, and
second to further characterize the type of damage which has taken
place.
The nucleoside analog 5-aza-20 -deoxycytidine (5-aza-CdR)
which is a potent inhibitor of DNA methyltransferase, is a drug cur-
rently in clinical trials for the treatment of solid tumors and leuke-
49
mia. Several laboratories including our own have published data
suggesting that 5-aza-CdR plays diverse roles in the cell which in-
clude both demethylating functions and methylation-independent
functions [6,7,22]. The cytotoxicity of this drug is potentially
attributable to several possible mechanisms. This particular study
was undertaken to test the hypothesis that 5-aza-CdR treatment
brings about DNA damage in the lung cancer cell line A549. In view
of the fact that there is a wide variety of cell type specific DNA
damage, the versatility of the alkaline comet assay for evaluating
DNA damage rendered the comet assay an ideal choice for this re-
search problem.
DNA damage in proliferating cells activates a pathway which
arrests cell division to allow either DNA repair or induction of cell
death by apoptosis. A549 cells under different doses and lengths of
time of 5-aza-CdR treatment were collected for comet assay. As the
type(s) of DNA damage which would result in this experiment
could not be predicted beforehand, the alkaline comet assay was
particularly useful, as it has the capacity to measure a mixture of
direct strand breaks and DNA damage (for example, SSB or ALS).
In this system, after determination that DNA damage had taken
place by use of the comet assay, the type(s) of DNA damage present
were further evaluated by flow cytometry and Western blotting
with anti-RPA (replication protein A) [23] or gamma-H2AX [6].
In this model the comet assay was also used as a rapid test to
investigate repair of DNA lesions after removal of the reagent 5-
aza-CdR from the medium. In a previous study we found 5-aza-
CdR acts in conjunction with the histone deacetylase inhibitor
(HDACI) depsipeptide to induce significant apoptotic cell death in
lung cancer cell lines [5], and we were thus led to consider whether
depsipeptide affects DNA repair enzymes to enhance 5-aza-CdR-
induced DNA damage. This hypothesis was also tested and verified
with use of the comet assay.
4.1. Detailed protocol for comet assay used in these experiments
The procedure for this assay is presented in Fig. 2. A549 cells
were treated with 5-aza-CdR at concentrations of 0.1, 1, and
5 lM for 72 h, and were then harvested. Fully frosted microscopic
slides were covered with 110 ll of 0.5% normal molten agarose at
60 °C. The slides were immediately coverslipped and kept at 4 °C
for 15 min to allow the agarose to solidify. Approximately 105 cells
treated with 5-aza-CdR or untreated in 40 ll of phosphate-buf-
fered saline were mixed with an equal amount (40 ll) of 1% lower
melting agarose to form a cell suspension. After gently removing
the coverslip, the cell suspension was pipetted onto the first aga-
rose layer, spread using a coverslip, and maintained at 4 °C for
15 min to allow it to solidify. After removal of the coverslip, the
slides were immersed in freshly prepared cold lysis solution
(2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10.0, 1% sodium
sarcosinate) with 1% Triton X-100 for 40 min at 4 °C. The slides
were then placed in a horizontal gel electrophoresis tank filled
with fresh electrophoresis solution (1 mM Na2EDTA, 300 mM
NaOH, pH 13.0) for 10 min (300 mA at 20 V). Following electropho-
resis at 4 °C, slides were rinsed twice in Tris buffer (0.4 M Tris, pH
7.5) for 15 min (neutralizing the excess alkali). The slides were
then stained with 75 ll of propidium iodide (5 lg/ml) for
30 min., and examined at 600 magnification. Pictures were taken
with a fluorescent microscope (TCS; Leica, Mannheim, Germany).
To score the percentage of DNA in the tail, an image analysis sys-
tem was used (Q550CW; Leica). The percentage of comet tail area
(DNA tail area/ area of total DNA) and the comet tail length (mea-
sured from the center of the DNA head to the end of the DNA tail)
were analyzed in 50 cells on one slide.
Using this protocol, the comet assay was used to test whether
depsipeptide affects removal of incorporated 5-aza-CdR, and a de-
cay analysis of incorporated [3H]-5-aza-CdR was carried out in
50 W. Liao et al. / Methods 48 (2009) 46–53

A549 cells. Cells were treated with [3H]-5-aza-CdR at 1 lM for

48 h, and with and without depsipeptide at 0.1 lM for the final
6 h of treatment, and were then washed and replaced in fresh med-
ium (without experimental reagents) for 6, 12 and 24 h.

4.2. Result analysis

Dose-dependent DNA damage was observed after 5-aza-CdR

treatment (Fig. 3). Compared with the untreated control (Fig. 3A),
5-aza-CdR-induced DNA damage even at a low concentrations
(0.1 lM), as indicated by the presence of the DNA tail (Fig. 3B). A
greater DNA tail area and longer DNA tail length (distance from
DNA head to the end of DNA tail) reflect more extensive DNA dam-
age. This evidence of 5-aza-CdR-induced DNA damage was ob-
served more frequently in cells treated with a higher
concentration of 5-aza-CdR than that with a lower concentration
of 5-aza-CdR, as shown by comparison of C and D with A (Fig. 3).
The quantitative comet assay data for 5-aza-CdR-induced DNA
damage are shown in Table 2. For example, the percentage of the
DNA tail area relative to the total area and the DNA tail length in-
creased 2.6- and 1.4-fold, respectively when cells were treated
with 5-aza-CdR at 0.1 lM as compared with that in untreated cells
(Table 2). Similarly, in cells treated with 5-aza-CdR at 5 lM, the

Table 2
Comparison of degree of DNA damage after 5-aza-CdR treatment using the comet
assay in A549 cells.

Treatment (lM) Tail area/total area (%) DNA tail length

Fig. 2. General schematic of the alkaline comet assay. The basic steps of the assay 0 17 ± 9 18.33 ± 2.1
which are illustrated here include single cell suspension, slide preparation, lysis of 0.1 44 ± 21 25.38 ± 3.5
cells to liberate DNA, exposure to alkali to obtain single-stranded DNA and to 1 58.3 ± 1 60.07 ± 10.2
express ALS as SSB, electrophoresis, neutralization of alkali, DNA staining, comet 5 70.6 ± 3 70.2 ± 15
visualization and scoring.

Fig. 3. Evaluation of 5-aza-CdR DNA damage by comet assay. DNA damage is calculated as the DNA tail area/whole DNA area (%) and the comet tail length (from the center of
DNA head to the end of the DNA tail) in A549 cells. The bigger the DNA tail area (%) or the longer the DNA tail length, the more significant the damage. (A) Control, the DNA is
intact without a DNA tail, (B) 5-aza-CdR, 0.1 lM, (C) 5-aza-CdR, 1 lM, (D) 5-aza-CdR, 5 lM.
 W. Liao et al. / Methods 48 (2009) 46–53 51

percentage and the length were increased 4.2- and 3.8-fold, respec- Table 3
tively (Table 2). These results suggest that 5-aza-CdR inhibits cell Recovery form 5-aza-CdR-induced DNA damage and inhibition of recovery by
proliferation by damaging DNA. DNA damage is characterized in depsipeptide were analyzed with the comet assay.

A549 cells as the DNA tail area/whole DNA area (%) and the comet Treatment Tail area/total DNA tail
tail length (from the center of DNA head to the end of the DNA tail). area (%) length
In Fig. 4, it is clear that 5-aza-CdR-induced DNA damage gradu- CTR 6.6 ± 2 2.2 ± 0.2
ally reversed when cells were cultured in 5-aza-CdR free medium. Depsipepide 6 h 6.9 ± 2 2.3 ± 0.2
The length and area of the DNA tail induced by 5-aza-CdR was 5-aza-CdR 24 h 40.2 ± 4 26.2 ± 2.0
5-aza-CdR 24 h + incubation 12 h 26.6 ± 4 9.8 ± 1.4
shorter and smaller when cells were cultured in 5-aza-CdR free
5-aza-CdR 24 h + incubation 24 h 14.8 ± 2 4.8 ± 1.2
medium for 12 h as compared to cells without a period of drug-free 5-aza-CdR 24 h + depsipeptide 24 h 45.4 ± 6 27.9 ± 3.2
incubation (Fig. 4A–C). This process of recovery from 5-aza-CdR-in- 5-aza-CdR 24 h + depsipeptide 24 h + 53.4 ± 6 27.9 ± 3.2
duced DNA damage was significantly suppressed by depsipeptide, incubation 12 h
so that the length and area of the tails in the comets were longer 5-aza-CdR 24 h + depsipeptide 24 h + 72.2 ± 14 70.1 ± 10.3
incubation 24 h
and larger then when cells were treated with 5-aza-CdR and dep-
sipeptide together (Fig. 4D–F). This depsipeptide-induced suppres-
sion of recovery from 5-aza-CdR-induced DNA damage is
summarized in Table 3. These data suggest that HDAC inhibitors
may suppress recovery from 5-aza-CdR-induced DNA damage aza-CdR-induced DNA damage. Moreover, Western blotting using
and synergistically induce inhibition of cell proliferation. anti-RPA (replication protein A) or gamma-H2AX showed that at
1 lM 5-aza-CdR did not induce DSB. However, 5-aza-CdR at higher
4.3. Combined use of other DNA damage assays for further analysis of concentration (5 lM) induced dose-dependent DSB. These findings
comet assay results together led to the conclusion that 5-aza-CdR induction of DNA SSB
or DSB is dose-dependent. Thus, the data from the comet assay was
Apoptosis also gives a positive result in the comet assay. We consistent with that from the other methods of assay.
therefore used flow cytometry analysis and Western blotting for
anti-PARP (cleavage of PARP is a hallmark of apoptosis) to deter- 5. Advantages and limitations of the comet assay, and compari-
mine whether 5-aza-CdR-induced DNA damage elicits apoptotic son with other assays for DNA damage
cell death. As shown in a previous study [6], 5-aza-CdR alone at
limited concentration (under 1 lM) is not able to induce apoptosis, The comet assay is a relatively fast, simple, and sensitive tech-
and thus the positive result in the comet assay is due mainly to 5- nique for the analysis of DNA damage in all cell types, and com-

Fig. 4. Depsipeptide inhibits removal of incorporated 5-aza-CdR from DNA. (A–C) The comet assay showed that 5-aza-CdR-induced DNA damage recovers when the treated
cells were incubated in 5-aza-CdR free medium for 12 h. (A) Untreated control. (B) 5-aza-CdR at 1 lM for 24 h without incubation, and (C) 5-aza-CdR at 1 lM for 24 h,
followed by washing for further incubation in a drug-free medium for 12 h (D–F). Recovery of DNA damage induced by 5-aza-CdR was inhibited by depsipeptide treatment
(0.1 lM for the final 6 h). (D) Depsipeptide treated cells (0.1 lM) for 6 h. (E) Cells were treated with 5-aza-CdR (1 lM for 24 h) and depsipeptide (0.1 lM for final 6 h) without
incubation, or (F) cells treated as above and further incubated in a drug-free medium for 12 h.
52 W. Liao et al. / Methods 48 (2009) 46–53
bines the simplicity of biochemical techniques for detecting DNA
SSB, ALS and cross-linking, with the single cell approach typical
of cytogenetic assays. Even though the comet assay has many mer-
its, at present neither this assay nor any other single assay is a
completely reliable biomarker for genotoxic effects in DNA dam-
age. As such, complementary techniques are strongly suggested
for study of the effects of genotoxic drugs. The following is a dis-
cussion of some other techniques available for DNA damage re-
search for comparison with the comet assay.
Analysis of the phosphorylation status of the minor histone
H2AX has been recognized to be a sensitive method for locating
DNA double-strand breaks, but the result is an average, rather than
a single cell assay [24]. Flow cytometry and image cytometry can
be used to identify cells containing this specific local modification,
called gamma-H2AX, and the sensitivity of this method is about
two orders of magnitude greater than that can be achieved by mea-
suring double-strand breaks using the neutral comet assay or
pulsed field gel electrophoresis [24,25]. However, in addition to
its single cell capacity, an advantage of the comet assay not found
it these other methods is that it is able to detect a wide variety of
additional types of DNA damage including single-strand breaks,
DNA interstrand crosslinks, and damage of bases that result in
endonuclease sensitive sites. In this regard, the versatility of the
comet assay is unmatched by other assays.
Under alkaline conditions, necrotic or apoptotic cells can result
in comets with small or nonexistent head and large diffuse tails
(called a ‘‘hedgehog” comet) [26] as observed in in vitro studies
following treatment with cytotoxic but nongenotoxic compounds
[27,28], However, ‘‘hedgehog” comet images must be interpreted
with caution in diagnosis of apoptosis or necrosis. For example,
treating cells with a non-lethal dose of an agent such as H2O2 re-
sults in comet images. However, if the treated cells are incubated
without H2O2 for 30 min and subjected to the comet assay, these
hedgehog comet are no longer seen, as cells have already repaired
the DNA breaks. In this situation, the hedgehog comets initially
observed cannot be interpreted as representing apoptosis, as
apoptosis is by definition irreversible [29]. In addition, the comet
assay is not particularly good for distinguishing apoptosis from
necrosis, [30,31] although measuring the degree of DNA fragmen-
tation can be helpful in this regard [32]. Flow cytometric analysis
has many advantages over use of the comet assay for measure-
ment of apoptotic cells in cases where extent of apoptosis is
the only piece of data being sought, as for example in our recently
published paper [40] where flow cytometric analysis was used to
evaluate apoptosis when cells were treated with depsipeptide.
Two approaches have been used for measuring oxidative DNA
damage which may be a cause of cancer. The most commonly
used biomarker for DNA oxidation is 8-oxo-7,8-dihydro-20 -deoxy-
guanosine(8-oxodGuo). Analysis for 8-oxodGuo by HPLC with
electrochemical detection can be performed on lymphocytes
and other cells. However, an alternative enzymatic approach is
based on digestion of DNA with formamidopyrimidine-DNA gly-
cosylase (FPG) to convert 8-oxo-7,8-dihydroguanine (8-oxoGua)
to apurinic sites, and subsequently measuring these sites as
DNA breaks using the comet assay. As the ESCODD (European
Standards Committee on Oxidative DNA Damage) [33] stated in
2005, this enzymatic approach (using the comet assay) has gener-
ally given to estimate of 8-oxoGua that are several times lower
than those obtained using HPLC, and cannot be used when the
extend of DNA damage is high. However, on the other hand there
is convincing evidence that substantial oxidation of guanine oc-
curs during preparation of samples for HPLC analysis, due to
the protracted and vigorous procedures that take place before
chromatographic analysis. The comet assay method is less likely
to suffer from spurious oxidation due to its minimal sample pro-
cessing requirements.
6. Conclusion
Both studies carried out in our laboratory as well as studies
published by other groups have demonstrated that the comet assay
is highly useful tool for measuring DNA damage in cells, and this
assay is widely used in fields ranging from molecular epidemiology
to genetic toxicology. However, because the extent of DNA strand
breaks will vary depending on the agent used to induce damage,
the nature of the lesions produced, and the type and biological con-
dition of cells or tissues, there has been observation of variability in
results from cell type to cell type, gel to gel, culture to culture, and
among various type of experimental animals. Even with adoption
of a standardized assay protocol, further work for standardization
of this assay is warranted. For example, a standardized statistical
method for the evaluation of comet data, which would allow direct
comparison of data from dose–response curves and levels of effect
of reagents under evaluation.
The standard comet assay reveals only strand breaks and alkali-
labile sites. In addition, there are other drawbacks such as the fact
that effects on cell-cycle checkpoints are not identifiable with this
assay, the data is single cell data (which requires evaluation of
many cells to give reliable quantitative results), small cell sample
(leading to sample bias), technical variability, and interpretation
are some of its disadvantages. Nonetheless, with judicious use,
the utility of the comet assay outweighs its potential drawbacks,
particularly when used in conjunction with other assays or in mod-
ifications chosen appropriately for the experimental design. There-
fore more widespread use of modified comet assays should be
encouraged.
Acknowledgments
This study was supported by Grant Nos. 30425017, 30670417,
and 30621002, 2005CB522403, 2006CB910300, and ‘‘111 project”
B07001.
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