Mutagenesis vol. 28 no. 3 pp. 333–340, 2013
Advance Access publication 5 March 2013
doi:10.1093/mutage/get012
High-throughput comet assay using 96 minigels
Kristine B. Gutzkow1, Torgrim M. Langleite2,
Silja Meier1,2, Anne Graupner1, Andrew R. Collins2 and
Gunnar Brunborg,1,*
Department of Chemicals and Radiation, National Institute of Public Health
(NIPH), PO Box 4404 Nydalen, Oslo N-0403, Norway and 2Department of
Nutrition, Institute of Basic Medical Science, University of Oslo, PO Box
1046 Blindern, N-0316, Oslo, Norway
1
*To whom correspondence should be addressed. Tel: +47 21076426; Fax: +47
21076686; Email: [email protected]
Received on April 2, 2012; revised on December 30, 2012; accepted on
January 9, 2013
The single-cell gel electrophoresis—the comet assay—has
proved to be a sensitive and relatively simple method that
is much used in research for the analysis of specific types of
DNA damage, and its use in genotoxicity testing is increasing. The efficiency of the comet assay, in terms of number
of samples processed per experiment, has been rather poor,
and both research and toxicological testing should profit
from an increased throughput. We have designed and validated a format involving 96 agarose minigels supported by
a hydrophilic polyester film. Using simple technology, hundreds of samples may be processed in one experiment by
one person, with less time needed for processing, less use of
chemicals and requiring fewer cells per sample. Controlled
electrophoresis, including circulation of the electrophoresis
solution, improves the homogeneity between replicate samples in the 96-minigel format. The high-throughput method
described in this paper should greatly increase the overall
capacity, versatility and robustness of the comet assay.
Introduction
Genotoxicity tests are essential for the pharmaceutical
industry and for regulation of chemicals within the European
Commission’s Registration, Evaluation and Authorisation of
Chemicals programme. Such tests should have high sensitivity
and specificity, but they should also be simple and efficient in
their performance. Single-cell gel electrophoresis—the comet
assay—has been shown to be suitable as a mammalian test for in
vivo and in vitro investigations of drug candidates and chemical
compounds (1–4). In addition, the comet assay has been highly
useful for analysing DNA damage and repair in large human
population studies (5–7), as well as for research in general.
The assay is highly sensitive for measuring DNA strand breaks
and may also detect specific DNA lesions by including DNA
repair enzymes, which recognise base oxidations (8). Several
protocols and versions are in use, mostly based on a glass
slide as substrate for one or two agarose cell samples, which
are spread out by means of a coverslip. With this technology,
more than 50 samples are, however, not easily handled in one
experiment because the processing of samples is manual and
time consuming. Both the application of gels and subsequent
handling of glass slides are susceptible to errors. The comet
assay in its traditional version, therefore, appears to be less
suitable for large screening studies in which a high-capacity
robust and standardised assay is needed. In addition, a method
requiring fewer cells and consequently lower amounts of the
drug or chemical compound to be tested may be useful. The
scoring of comets represents a very time-consuming step; when
revising the technology, the possibilities for automation should
be addressed.
We have devised, and described in this report, a modified
comet assay based on applying small droplets of agarose with
cells (minigels) onto a polyester film (e.g. GelBond®). Using
simple technology including a modified electrophoresis system,
hundreds of samples can be processed per experiment by one person. The various steps are suitable for further automation, and the
format can be adapted to fully automated scoring. The assay gives
results that are indistinguishable from the traditional comet assay.
Materials and methods
Isolation of human peripheral blood lymphocytes
Human peripheral blood lymphocytes (HPBL) were isolated from whole blood
or buffy coat obtained from healthy consenting volunteers, using standard
Ficoll–Hypaque density gradient according to the manufacturer’s protocol
(Lymphoprep™, Axis-Shield PoC AS, Oslo, Norway). Typical yields were
1–2 × 106 cells/ml blood.
Aliquots of HPBL were prepared and used fresh. Alternatively, they
were frozen (unexposed or after irradiation) for later use for convenience
and to allow comparison of samples in different experiments. Cell samples
were pelleted and resuspended in freezing medium [RPMI 1640 with 2 mM
l-glutamine, 20% fetal bovine serum and 10% dimethylsulphoxide (DMSO)]
to a concentration of 2 × 106 cells/ml. Slow freezing was performed by placing samples in an isopropanol-filled insulated box (Mr Frosty; Nalgene Cryo,
Thermo Fisher Scientific, Germany) which was then placed in a −80°C freezer.
For use, samples were quickly thawed by adding 1 ml of cold phosphatebuffered saline (PBS) to the partly thawed cell suspension before tipping the
contents into 10 ml of cold PBS. Cells were pelleted by centrifugation at 400
× g for 10 min, and the titre adjusted to 0.5–1.0 × 106 cells/ml in PBS prior to
mixing with agarose for comet analysis.
Genotoxic treatment of cells
To induce DNA strand breaks and alkali-labile sites, HPBL were irradiated in
tubes placed in a Petri dish filled with ice/water, with X-rays (0.1–15 Gy) delivered by a Phillips Model MG300 X-ray unit (Germany) operated at 260 KeV
and 10 mA, or by a PXI XRAD225 unit (225 KeV, 13 mA). Radiation was
filtered through 0.5-mm copper. The dose rate with copper filter was 10 and 3
Gy/min for the Phillips and the PXI units, respectively, as determined with iron
sulphate dosimetry (9). Irradiated cells were kept ice cold, except during rapid
mixing with agarose and casting, followed by transfer to the lysing solution
without delay. When specified, cells were irradiated after embedding, either on
glass slides or plastic film kept ice cold. Unexposed samples were lead shielded.
To induce DNA lesions detectable by the enzyme formamidopyrimidineDNA glycosylase (Fpg) (‘Fpg-sensitive sites’, mainly 8-hydroxy-7,8dihydroguanine, 8-oxoG), freshly prepared HPBL were treated with the
compound Ro 19-8022 plus visible light. Ro 19-8022 dissolved in DMSO
(stock kept at −20°C) was added at 1:400 to a final concentration of 1 μM
(0.25% DMSO was added to control samples), in dim light. After 1 min, the cell
suspension, placed on ice, was exposed for varying periods of time to visible
light from a 500-W halogen source, at an intensity of 35 000 lux at a distance of
30 cm. Cells were then pelleted for 7 min at 400 × g at 4°C. The supernatant was
removed, and the pellet was gently resuspended (using a large-bore pipette tip)
and washed in 10 ml of cold PBS before finally resuspending at a concentration
of 1 × 106 cells/ml. Samples exposed for different periods of time were analysed
in the comet assay.
Making the minigels
Several gel formats were explored (Figure 1). Cells were gently resuspended
and mixed in the ratio of 1:10 with 0.75% (final concentration 0.67%) lowmelting point agarose (NuSieve® GTG® Agarose, Lonza, Rockland, ME,
© The Author 2013. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
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K. B. Gutzkow et al.
Fig. 1. The traditional and modified comet assay formats. Top: the glass slide format. Below: various formats using hydrophilic polyester films as substrate: the
12-gel format (for which a brass/Teflon mould is used), the medium- and the high-throughput versions.
USA) in PBS (pH 7.4) without calcium and magnesium and with 10 mM
Na2EDTA. Aliquots were then added to glass or polyester film substrates as
described in the below section. The traditional comet assay involves embedding
cells in a flat agarose gel on a glass slide, achieved by covering the agarose/cell
droplet with a coverslip; this protocol has been described previously (10) with
modifications (8,11).
For a polyester films as substrate, pre-cut sheets (GelBond®) were used, each
with 5-mm holes punched in its corners so that the film could be hooked onto
a plastic support frame via four stainless-steel legs (Figure 2). This stretches
the film more or less flat; the frame also facilitates subsequent handling and
protects the gels from mechanical damage. Film sheet sizes were 78 × 110 mm
(12 or 48 samples), or 85 × 125 mm (96 samples) (Figure 1).
For both formats (48 and 96 samples), cell samples were mixed with agarose—using an 8-channel pipette—in microcentrifuge tubes or a microtitre
plate on a heating block at 37°C. After gentle mixing, samples of the cell/agarose mixture were added as 7 μl (6 × 8 = 48) or 4 μl (8 × 12 = 96, spaced as in a
standard 96-well microwell plate) to each film using an 8-channel pipette (for
>4 µl an 8-channel pipette with adjustable tip distance was used). To ensure
even spacing of samples, a master plastic plate with conical holes for the tips
was used. (As a simple but less precise alternative, a white paper with the positions indicated may be placed underneath the film.) When applying the agarose
samples with the pipette, the tips should touch the film surface at a slight angle
(70°); it takes very little training to master this skill. The agarose/cell mixtures
were added at a gentle flow, preferably without moving the tips and with no
swirling. This procedure results in minigels that are circular, regular in size and
of similar thickness. The films were immersed in cold lysing solution once all
samples had been added (1–8 min).
Processing of embedded cell samples
Lysis took place at 4oC (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% sodium lauryl sarcosinate, to which 1 ml of Triton X-100 and 10 ml of DMSO per 100 ml
solution had been added the same day; final pH 10) for 1–18 h (for most—but
not all—types of DNA lesions, the duration is not important). Films/glass slides
were then either transferred directly to electrophoresis solution for unwinding
or an enzyme treatment step was included. For the latter, films were washed
with 1 × 10 min plus 1 × 50 min in enzyme buffer (40 mM HEPES, 0.1 M KCl,
0.5 mM EDTA, pH 8.0) at 4°C. Fpg, isolated from a bacterial strain containing
an over-producing plasmid (12), with modifications (13), was added at 1 µg/ml
(or no enzyme for control films) to enzyme buffer with bovine serum albumin
(0.2 mg/ml) at 37°C. Films were then immersed and incubated in this solution
for 1 h at 37°C. For samples on glass slides, washing in buffer was followed by
addition of 45 μl of the Fpg diluted 1/3000 to each sample and incubation for
30 min at 37°C (14). The enzyme preparations used for glass slides and films
were different extractions but obtained from the same clone. For both preparations, the treatment conditions (enzyme concentration/time) were tested to
establish first-order reaction kinetics.
Unwinding and electrophoresis
For unwinding, films/slides were immersed in cold electrophoresis solution
(0.3 M NaOH, 1 mM Na2EDTA, pH > 13.2) for 5 min and then for another
35 min in fresh solution. Electrophoresis was carried out in cold, fresh electrophoresis solution (8–10°C) for 20 or 30 min as specified. Circulation of
the electrophoresis solution in the electrophoresis tank was employed when
334
specified (75 ml/min using a peristaltic pump). For glass slides, the nominal
electrophoresis voltage (of the power supply) was 20 V, and the electrophoresis solution was adjusted so that the current was ~300 mA. A larger tank was
designed to accommodate four 96-format films; in this tank, electrophoresis
took place at 22.5 or 25 V (as specified) nominal voltage; the power supply
was custom-made from two serially connected lead sulphate batteries plus a
voltage regulator. This gives a stable DC voltage, and there is sufficient power
for at least three electrophoresis tanks to be run in parallel (i.e. 1152 samples in
total). With 1.4 l of electrophoresis solution in one tank, the depth of the liquid
on the platform was 9 mm, and the current was 700–800 mA. For both types
of tanks, the voltage potential (which is the driving force in electrophoresis of
charged molecules such as in the comet assay) (15) was 0.8–0.9 V/cm on the
platform, where the samples are placed. Following electrophoresis, slides/films
were immersed in Tris buffer (0.4 M Tris, pH 7.5) for 2 × 5 min for neutralisation, rinsed in distilled water, followed by fixation in 96% ethanol for 1.5 h
and then drying overnight. Films treated in this way may be stored for at least
1 year before rehydration and staining.
Scoring of comets
For staining, slides/films were rehydrated in TE buffer (10 mM Tris–HCl, 1 mM
Na2EDTA pH 8.0), containing SYBRGold (Life Technologies Ltd, Paisley,
UK) at 10 000× dilution, for 20 min at room temperature with mild shaking.
Thereafter, slides were quickly rinsed in distilled water, covered with large coverslips (80 × 120 mm, thickness no. 1; VWR International AS, Oslo, Norway)
and stored in a dark, moist chamber at 4°C until scoring the same or the next
day. For semi-automated scoring, the software Comet assay IV (Perceptive
Instruments Ltd, Bury St Edmunds, UK) was used, with either Leica DMLB
(light source Osram Mercury Short ARC HBO® 50W/2) or Olympus BX51
(Osram Mercury Short ARC HBO 100 W/2) epi-fluorescence microscopes
equipped with ×20 lenses. Fifty comets were scored per gel. Care was taken to
avoid selection bias, that is, overlapping—or potentially overlapping—comets
were excluded. Because the minigels contain relatively few cells, the cell density is important and should be <1200 cells in a 4-μl sample, corresponding to
3 × 106 cells per ml prior to dilution and embedding in agarose.
Statistics
Median (or mean, when specified) values were calculated from populations of
individual comets (% tail DNA). Linear regression lines (slopes) were fitted to
these data (or means of parallel samples) to construct dose-response curves;
the mean ± SD of the slopes of these curves were calculated. For the low-dose
irradiation experiment (Figure 6), two-sample non-parametric test (Mann–
Whitney U-test) was used to test for significant difference between groups
(population of comets). Figure and table legends define further statistical
comparisons. Excel 2010 and IBM SPSS Statistics 20 were used for statistical
calculations.
Results
Plastic films as substrate for agarose gels
The use of polyester plastic films such as GelBond® as substrate
in the comet assay was first described by McNamee et al. (16)
High-throughput comet assay
Fig. 2. The 96-format film. Left, a polyester film attached to a plastic frame with metal hooks in each corner. Also shown is the application master with 96 holes
used for precise positioning of the gels. Right, a film with 96 regular minigels.
and has later been applied by several investigators (17,18) but
otherwise, the protocols of these investigators were generally in
accordance with the standard comet assay protocol (19). With
its hydrophilic surface on one side to which the agarose sticks
firmly, polyester films of various sizes have been used in our
laboratory as substrate for different comet formats (Figure 1).
With the 12-gel version, 50–100 μl are applied using moulds
giving circular gels of even thickness (20,21). The smaller
(minigel) samples are obtained by application of 6 × 8 or 8 × 12
gels (of equal size and spacing) to a film, using an 8-channel
pipette (Figure 2). With the described procedure, the agarose
samples—once added to the film surface—spread out readily
forming round, thin lens-shaped gels of equal size, which
solidify within seconds allowing immediate lysis. This rapid
processing contributes to the prevention of unspecific DNA
cleavage as well as DNA repair at elevated temperatures.
The various formats used as illustrated in Figure 1 are
obtained using simple devices that any instrument workshop
can make. A cutting device (made from a paper cutter with
guides attached) produces films of correct size. Two modified
office paper punches are used to make holes in each corner. As
shown in Figure 2, the films are hooked onto plastic frames with
stainless-steel metal feet (custom-made), before application of
gel samples. After application, the samples are protected by the
frame against mechanical damage; their handling between the
different treatment steps is quick and safe. The 96 samples are
positioned using an application master (Figure 2). A pipetting
robot may be used to achieve the high precision needed for fully
automated scoring. For scoring, the films may be cut to any
size either before or after staining, and they are then supported
on a glass surface. As an alternative, we devised a microscope
stage that can take one complete film with a large coverslip
covering its entire surface. The film format is suitable for fully
automated scoring (22) although more validation is needed.
Time needed for processing
The time needed to process samples in the comet assay has
been drastically reduced using the minigel format (Table I);
the increased speed is particularly related to a more efficient
sample mixing and application to the substrate. With little
training, an operator can make one film with 96 different
samples in 4–8 min (starting with samples ready to be mixed
with agarose) using an 8-channel pipette; four parallel films
used for enzyme treatment are completed in <22 min. In
comparison, it takes an experienced operator approximately
6 min to apply 10 samples to 10 standard glass slides (two
duplicate samples per slide) (Table I). These estimated times
are for comparison and include the time needed for preparations
such as pre-coating glass slides (or cutting films), labelling,
mixing cells with agar, adding and removing coverslips etc.,
and also preparations for electrophoresis and staining (but not
the duration of electrophoresis itself). Overall, with the highthroughput 96-minigel format comet assay, samples may be
processed through the relevant steps approximately 10 times
faster (per sample) than with the traditional glass slide method
(scoring not included).
Minigel flatness, thickness and swelling
During validation, various factors that could affect the comet results
were investigated. For scoring, especially in a fully automated
system, it is important that the comets lie in approximately the
same focal plane, which depends on the flatness and thickness
of the gel. Minigels made without coverslips are lens shaped,
with maximum thickness of approximately 0.06 mm compared
with a standard flat gel of 50 μl with more uniform thickness of
0.03–0.15 mm (size 300–1200 mm2). These data were obtained
by means of a microscope and focusing on cells at the bottom
or near the top of minigels, after rehydration and staining. (The
thickness is reduced during drying, but the minigels swell to
30–80% of their original thickness during rehydration and
staining.) The corresponding focal plane and gel thickness are
suitable for semi-automated or automated scoring.
If minigels are left at room temperature for more than a few
minutes after application, they may dry along their periphery
resulting in apparently longer comets; this was not a problem
when keeping the film on a cold support followed by rapid
immersion into the lysing solution (data not shown).
Number of cells per minigel
The number of cells contained in one gel was varied in order
to establish optimal numbers. When the initial concentration
of the cell suspension exceeded approximately 3 × 106 cells/ml
(i.e. >1200 per 4 µl of gel), the scoring became increasingly
difficult due to many overlapping comets particularly at high
levels of damage. At or below 1200, the measured median %
tail intensity of HPBL irradiated with 10 Gy was close to results
obtained with 200–400 comets per gel (results not shown). An
optimum cell concentration was, therefore, established as 100
cells per µl minigel. Lower cell numbers can be used at the
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K. B. Gutzkow et al.
Table I. Processing times in the minigel and the traditional method Comet assay versions: minigel
method/traditional method
Minigel method
Traditional method
Process
One film 96 samples
(seconds per film)
One film 96
samples (seconds
per sample)
Four parallel films,
384 samples in total
(seconds per sample)
Two samples per slide
(seconds per sample)
Cutting film/pre-coating slides
Mixing cells with agarose
Adding samples to substrate
Adding and removing coverslips
SUM processing time until lysis
Preparation for electrophoresisa and
transfer of samples between solutions
(4 films or 16 slides)
Preparation for stainingb (4 films or
16 slides)
SUMa,b
Comment
60
120
300
0
480
120
0.6
1.3
3.3
0
5.2
1.2
0.6
1.3
1.5
0
3.4
0.4
5
5
15
3
28
4
60
0.6
0.3
4
660
7
4.1
Fewer cells needed, less chemicals and agarose per sample, cost per sample is
reduced by 50–150% (chemicals not included)
36
Versatile; useful for
small experiments
Approximate times are shown for one film with 96 samples, per one sample and per one sample when making four parallel films. Estimated times are also shown
for the traditional glass slide method. Italics: type of assay; bold: sum of the specific processing times.
a
Excluding actual electrophoresis time.
b
Excluding actual staining time.
Fig. 3. Electrophoresis of 96 minigels in a modified electrophoresis tank. X-irradiated (10 Gy) and non-irradiated lymphocytes in an array covering the entire
film (A) were subjected to electrophoresis with or without circulation (75 ml/min) at 25 V (0.9 V/cm) for 20 min. The figure (B) illustrates variations in the
response of individual samples (10 Gy) with and without circulation. Data pooled from three separate experiments. Calculated mean levels of DNA damage are
shown in Table II.
expense of the time needed to locate and score them. All cells
in a population of approximately 50 were traceable in a 4-µl gel
(using ×5 lens; results not shown).
Optimising electrophoresis
Custom-made electrophoresis tanks, each holding four films,
were designed. An increased volume of the electrophoresis
solution (total depth 8–9 mm) makes the system less sensitive
to varying depths of the solution on the platform, which is often
seen when the depth is 1–3 mm and the tank is not accurately
levelled. Sufficient current is supplied by two serially connected
lead accumulators with integrated meters to monitor voltage
and currents as described in Materials and methods. Using
this device, at least three electrophoresis tanks with four films
336
each, may be run in parallel at a constant voltage of 25 V. The
voltage drop across the platform may be controlled with probes
connected to a standard digital voltmeter. Higher precision
is obtained using a measuring gauge equipped with platinum
probes at defined height and spacing. With this device, we
have recorded local variations in voltage during a run; such
variations are reduced by mild circulating of the solution via an
external pump (data not shown).
Homogeneous response of neighbouring samples in
electrophoresis
X-ray-treated (10 Gy) and non-treated normal human
lymphocytes were added to films as illustrated in Figure 3,
to achieve a distribution of damaged and undamaged comets
High-throughput comet assay
across the area. The mean comet score of the 10 Gy samples
among replicate gels across the film—electrophoresed in the
large tank with 8–9 mm depth—showed a substantial variation,
which was considerably lower when the solution was circulated
(Figure 3 and Table II). In addition, the average damage
response of 10-Gy-treated samples was substantially higher
when circulation was included.
Comparing DNA damage response measured with the
minigel versus the glass slide method
The validation included a series of experiments ensuring that
the minigel method gives results quantitatively comparable to
the glass slide method. HPBL were exposed to various doses
of X-rays, either in suspension or after embedding in agarose.
The resulting dose-response curves in Figure 4 were similar for
minigels on film and gels on glass slides (the relative difference
in their slopes was 18 and 6% for irradiation in suspension
and in gels, respectively). For glass slides, the slope was 14%
higher for embedded cells than for cells in suspension, whereas
for minigels, the slopes were almost identical irrespective of
the mode of exposure (3% difference). This suggests that the
slower processing of samples for glass slides after irradiation
may allow some repair of induced DNA damage to take place.
To verify stable electrophoresis conditions across the film surface at varying damage levels, samples of HPBL irradiated with
increasing X-ray doses were positioned in alternating directions,
in 12 lanes, on two different films. Figure 5 shows linear dose
responses and associated regression parameters. The 12 lanes
gave highly similar dose-response curves, with coefficients of
variance (CV) = 3.1 and 6.1% for Film 1 and Film 2, respectively.
The comet assay is sensitive to very low levels of γ radiation. Using standard glass slides, Tice and co-workers (23,24)
showed that doses as low as 0.05 Gy gave significant changes
in the distribution of comet tail lengths in samples of HPBL.
Figure 6 shows a similar experiment using minigels for the
analysis of HPBL exposed to X-rays. The electrophoresis time
was increased from 20 to 30 min for increased sensitivity at low
doses (15) (electrophoresis for 40 min was used in (23,24)).
The response was linear and even the lowest dose (0.1 Gy)
produced a significant change (P = 0.017) in the population of
comets (Mann–Whitney U-test).
Validation of the high-throughput comet assay with lesionspecific enzyme treatment
The shape and thickness of minigels may affect the transport
of large molecules (proteins) during the enzyme treatment
Table II. Electrophoresis of minigels with or without circulation of the electrophoresis solution Cells irradiated with X-rays (10 Gy)
Experiment 1
Experiment 2
Experiment 3
With circulation
Without circulation
Mean of 12 replicates
(% tail DNA)
SD of 12 replicates
(CV, %)
Mean of 12 replicates
(% tail DNA)
SD of 12 replicates
(CV, %)
51.1
49.2
41.2
3.98 (7.79)
3.95 (8.03)
2.35 (5.70)
36.5
34.3
21.3
5.74 (15.73)
11.36 (33.12)
6.08 (28.54)
A cell sample was exposed to 0 or 10 Gy; from both exposures, 12 parallel samples were evenly distributed across the film, as shown in Figure 3A. The films
were subjected to electrophoresis at 25 V (0.9 V/cm) for 20 min, either with or without circulation. The mean value (% tail DNA) of 50 comets scored in the 10 Gy
samples was calculated for each sample. Mean, SD and CV (%) of the 12 parallel samples are shown, for each of three separate experiments.
Fig. 4. Comparing 96 minigels on films with standard gels on glass slides. Cells were irradiated with X-rays (0–15 Gy) on ice, either after embedding in gels
(right graph) or in suspension (left graph) followed by embedding of cells in agarose without delay. Films and glass slides were then quickly transferred to lysis
solution. Circulation was used during electrophoresis for the 96-minigel format (0.9 V/cm), whereas no circulation was used for glass slides (0.9 V/cm) in a
smaller tank. In both cases, electrophoresis was run for 20 min. Symbols denote the mean of two independent experiments in which 150 comets (50 × 3 replicates)
were scored for each sample in each experiment. Linear regression lines with slopes and R2 (goodness of fit) are given for each experiment. Dotted lines: minigels
on plastic film; solid lines: standard gels on glass slides.
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Fig. 5. Dose effect curves across the 96-minigel film. Lymphocytes in
suspension were irradiated with X-rays, and samples (0–15 Gy) were placed
across each of two films in 12 lanes, with increasing doses in alternating
directions. Electrophoresis was at 0.7 V/cm (with circulation) for 20 min.
Symbols denote DNA damage (means ± SD) of 12 replicate samples, each
with 50 comets scored. Linear regression lines with slopes, constants and R2
(goodness of fit) are given for mean of the dose response curves derived from
each film.
Fig. 7. Analysis of oxidative lesions using Fpg. Lymphocytes were treated
with 1 µM Ro 19-8022 plus visible light for 90, 180 or 270 s. Treated cells
were embedded in agarose and analysed as minigels (48 minigels with 7 μl
per sample), or using the standard glass slide method (results from two
separate laboratories). Electrophoresis was run at 0.9 V/cm for 20 min. DNA
damage is shown for samples with or without Fpg treatment (solid or dotted
lines, respectively). The data represent means ± SD calculated from three
independent experiments, each comprising three technical replicates.
assay included either immersion of films (48 × 7-μl minigels on
film) in a bath containing an Fpg extract, or adding droplets
of the extract to gels (glass slide format). Using both formats,
DNA damage in Fpg-treated samples increased linearly with
the visible light exposure (Figure 7). The results suggest no
difference in the efficiency of Fpg to detect and cleave the
induced DNA lesions.
Discussion
Fig. 6. Response to low doses of X-rays. Lymphocytes in suspension were
irradiated with X-rays (0–0.7 Gy) and samples were analysed in triplicates
using the 96-minigel format. Electrophoresis was run at 25 V (0.9 V/cm) for
30 min. DNA damage for populations of 150 comets are shown, with error
bars representing the standard error of the mean. The values for all irradiated
cells are significantly different from control (P = 0.017 or lower). Data are
from one representative experiment out of three experiments.
step, which is commonly used in the comet assay to detect
specific DNA lesions. Lymphocyte samples were treated with
the photosensitiser (Ro 19-8022) plus visible light to induce
oxidative DNA lesions (mainly 8-oxoG). The subsequent comet
338
Recently, a high-throughput assay was described (25), allowing
growth, chemical treatment and processing of cells for comet
analysis in microtitre wells, whose walls can be separated from
the plate. The technology was later adapted to non-adherent
cells including lymphocytes (26), and the authors combined
this system with fully automated scoring of comets (27).
Various chemical treatments were used to assess and compare
the performance of this system with the traditional comet
assay. Technical complexity may have limited a wider use of
this method because very few citations have been identified
until now. Ritter and Knebel (28) developed a system based on
20 samples spotted onto glass slides and an automated image
acquisition/image analysis to measure comet tail parameters.
Increased number of samples per glass slide has also recently
been described by Zhang et al. (29) and by us (30).
The minigel design described in this paper was validated
using X-rays because ionizing radiation is known to induce
defined levels of DNA strand breaks/alkali-labile sites per cell,
and X-rays are hence preferable for validation and calibration
purposes. Our results show that the minigel comet assay format
using a polyester film as substrate has the same dynamic range
for detecting DNA damage as the standard assay based on glass
slides, and the linearity of the dose-response curve is at least as
high (Figure 4).
Each minigel sample contains a sufficient number of cells
to allow satisfactory statistics. The opportunity to analyse very
High-throughput comet assay
low cell numbers may also be useful when studying cell types
such as oocytes or samples retrieved in biomonitoring studies.
The polyester film itself offers several advantages. No pre-coating prior to sample application is needed; the films are robust
and do not break, gels never fall off and gels may–if needed–be
applied or moulded in different sizes and formats. The plastic
film cannot, however, be used with all fluorochromes because
there is some autofluorescence of the GelBond® film in a wavelength region, which corresponds to the DAPI stain. Of the
fluorochromes we have tested, SYBRGold gives a very strong
signal with relatively little bleaching during scoring or between
scorings several weeks apart (data not shown) and anti-fade
is not needed. We use this fluorochrome to score rehydrated/
stained samples, which are either wet (i.e. with coverslip) or
semi-dry (no coverslip, dried for some hours/days).
The minigels are of similar thickness to a standard gel on
glass slide although they are far from flat. There is no apparent
effect of the lens-shaped minigel on the level of DNA damage, versus the level detected in the standard flat gel. An agarose gel consists of more than 99% water and would therefore
be expected to have similar electrophoretic properties as the
electrophoresis solution. There is also no indication of different
levels of DNA damage in different zones of the minigel (data
not shown).
With the increased electrophoresis time used to obtain the
results in Figure 6, the sensitivity of the minigel method using
polyester film as substrate is the same as for glass slides (23,24).
We have recently described how the dynamic sensitivity range
of the comet assay may be adapted by altering parameters such
as the electric potential (V/cm), the electrophoresis time and
the agarose concentration (15). A careful control of the electric potential during electrophoresis seems highly important to
reduce inter-experimental variability. Variations between parallel gels is likely to be more important if the gel sample covers a
large area; the 96-minigel format was useful to assay for local
in-homogeneities in DNA damage (reflecting, e.g. variations
in the electric field) across an electrophoresis tank platform.
Circulation of the alkaline solution during electrophoresis is
recommended because we found that it reduced the variability between parallel minigels across the same film (Figure 3
and Table II). Circulation gave a slightly higher median level of
DNA damage in the exposed samples, which is likely to reflect
that the electric potential close to a surface is affected by the
circulation.
The high-throughput minigel comet assay format appears to
work with any cell type or tissue and is more suitable than the
conventional method when large numbers of samples need to
be processed. Protocols for freezing and later analysis of large
numbers of cell/nuclei samples will contribute to a more versatile comet assay for in vivo testing (31). We have applied the
high-throughput minigel format for analysis of DNA strand
breaks and oxidative DNA lesions, in various types of tissues
from mice, such as the lung, spleen and brain (unpublished
data), testis and liver (21), and human sperm (20). Cells from
different species have also been tested with the new format
including fish, spring tails and sea stars (32). The minigels are
particularly useful when testing chemicals at multiple concentrations for genotoxicity. We obtained similar dose responses
of methyl methanesulphonate-treated cells when analysed
with the minigel format as with conventional glass slides (22).
Other examples of application of the method in the past for
analysis of genotoxicity are benzo[a]pyrene (20), acrylamide
and glycidamide (21), and nanoparticles (33).
Various modifications of the comet assay have appeared, which
aim at increased sample throughput, using single cells in an array
(34), in situ electrophoresis (25,26) or other formats (28). The format, which is described in this study, is highly versatile and flexible and may be used to analyse DNA damage in a variety of cell
types and tissues. The number and size of minigels may be varied,
using volumes of 3–7 µl. Larger gels can be made using moulds.
Commercial 96-format alternatives are available using glass
(Trevigen CometSlide™), but the plates are relatively expensive
and have to our knowledge not been validated. Our format based
on polyester (GelBond®) film uses simple technology, the design
is non-proprietary and relatively inexpensive (€1 per 96-format
film). The films are unbreakable and may be stored securely
requiring little space, for later re-analysis or for documentation.
The scoring of comets now remains as the time-limiting
step of the assay. Commercial systems are available allowing
fully automated scoring. We have compared automated, semiautomated and visual scoring of samples in various formats,
(22) and a further validation of the 96-minigel format on plastic
films is currently ongoing.
Funding
The European Commission projects Newborns and Genotoxic
exposure risks (NewGeneris; contract FOOD-CT-2005016320-2) and Comet assay and cell array for fast
and efficient genotoxicity testing (COMICS; contract no.
LSHB-CT-2006–037575).
Acknowledgements
The authors acknowledge Jan Zahlin for his contributions to the development
of technical prototypes and Michal Stawarski and Hildegunn Dahl for their
valuable technical help.
Conflict of interest statement: G.B. and A.R.C. have shares in two Norwegian
companies (CometBioTech and NorGenoTech) offering services based on
geno­toxicity testing and development, including the comet assay.
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