The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.
Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the “scoring” of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.
Cells are exposed continually to agents arising from the internal and external environments, which can damage DNA1,2. This damage can cause aberrant cell function3, and therefore DNA damage may play a critical role in the development of many major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging4. The comet assay (also called single-cell gel electrophoresis) is an increasingly popular method for detecting and quantifying cellular DNA damage.
At its simplest, the alkaline comet assay (ACA) detects strand breaks (SB; both single and double), together with apurinic/apyrimidinic sites and alkali-labile sites (ALS) both of which become single-strand breaks under alkaline conditions5. The neutral pH comet assay can evaluate frank single- and double-strand breaks6. Furthermore, the ACA, in combination with a number of DNA repair enzymes, can detect a considerable range of types of DNA damage, e.g., oxidized purines (identified by the use of human 8-oxoguanine DNA glycosylase 1; hOGG17); oxidized pyrimidines (using Endonuclease III; EndoIII) and cyclobutane pyrimidine dimers (using T4 endonuclease V; T4endoV)8. The comet assay can also be used to evaluate DNA lesions induced by crosslinking agents, such as cisplatin9,10,11. As indicated by the assay's formal name, i.e., single cell gel electrophoresis, the assay relies upon the cells under analysis being a single cell suspension; most commonly, these are cultured cells but may be isolated from whole blood12,13, or whole blood itself can be used14,15. Alternatively, a single cell suspension may be generated from solid tissues.
Apart from a few exceptions, most notably the CometChip reports from the Engleward lab16, the overall comet assay protocol has not changed dramatically from that originally described by the assay's inventors (Östling and Johansson17 and Singh et al.18). The comet assay involves numerous steps (Figure 1). Many of these steps involve the transfer of the thin, cell-containing agarose gels, one slide at a time, and, therefore, pose a risk of damage or loss of the gel, jeopardizing the experiment's success. Consequently, the comet assay can be time-consuming, particularly if a significant number of slides are being run. Typically, a maximum of 40 slides are run in a large (33 cm x 59 cm x 9 cm) electrophoresis tank, which sits within an even larger tray containing wet ice for cooling. It has been recently reported that the assay runtime can be shortened to 1 day by decreasing the duration of the lysis step and not drying the slides before staining19.
The present authors have previously reported a novel approach to the high throughput alkaline comet assay (HTP ACA), in which multiple (batches of 25) comet assay microscope slides can be manipulated simultaneously throughout the comet assay process20,21,22. This patented approach minimizes the risk of damage to, or loss of, the sample-containing gels by removing the need to manipulate the microscope slides individually and can be applied to all variants of the comet assay, which use microscope slides. The slide-containing racks protect the gels during the manipulations, and consequently, the sample processing is quicker and more efficient. The slides can also undergo electrophoresis in the racks, held in the vertical, rather than horizontal, orientation. This, and integral cooling, significantly decrease the footprint of the electrophoresis tank and removes the need for wet ice. Taken together, this represents a significant improvement over the conventional procedure. The equipment used is illustrated in Figure 2. The protocols described here, using this novel approach, demonstrate the representative application to cultured cells and whole blood14 for detection of alkali-labile sites (ALS), DNA inter-strand crosslinks (ICL), and the substrates of various DNA repair enzymes.
Commercially available blood samples were used in the present study. At our institution, Institutional Review Board approval is not needed for the use of commercially available blood.
1. Preparation of materials for the comet assay
2. Preparation of samples
3. Cell lysis
NOTE: Carry out all the procedures on ice.
4. Electrophoresis
5. Propidium iodide (PI) staining
6. Enzyme-modified alkaline comet assay
NOTE: The enzyme-modified alkaline comet assay employs an enzyme treatment step after lysis but before electrophoresis. The activity of the enzyme causes breaks in the DNA at sites that are substrates for the enzyme. Before performing this assay, enzyme concentration and duration of enzyme incubation must be optimized.
7. DNA inter-strand crosslinks (ICL)-modified alkaline comet assay
NOTE: The concept of this variant of the ICL-ACA is that the presence of ICL in DNA will retard the electrophoretic migration of damaged DNA, induced following exposure to an oxidatively generated insult. In this instance, the shorter the comet tail, the greater the number of ICL25,26,27,28.
8. Comet scoring and data analysis
NOTE: The term "comet" derives from the images of damaged cells when viewed under a microscope after the assay has been performed (Figure 5). Under electrophoresis conditions, DNA in the undamaged cells largely does not migrate, but remains in a spheroid termed as comet "head". However, the presence of strand breaks allows the cell's DNA to migrate out of the head, and form a "tail", thus leading to an appearance like a comet (Figure 5). The more DNA in the tail, the more damage is present.
Optimization of the electrophoresis voltage for the HTP ACA
Human keratinocytes (HaCaTs; Table of Materials) were irradiated with different doses of ultraviolet A radiation (UVA) (5 or 10 J/cm2; Figure 6A), UVB (0.5 or 1 J/cm2; Figure 6B), or treated with 50 µM H2O2 (Figure 6C) to induce damage. Three different voltages of the electrophoresis were tested to determine the optimal voltage for electrophoresis. The results from all three DNA damaging treatments revealed that, while all voltages generated linear dose-responses, the most sensitive response was obtained with 1.19 V/cm. HaCaTs showed the highest baseline DNA damage using 1.19 V/cm during electrophoresis compared to 1 V/cm and 1.09 V/cm (Figure 6A-C). In addition, using 1.19 V/cm, the greatest % tail DNA is seen, following all damaging treatments (Figure 6)31.
Detection of DNA damage in human whole blood using Fpg modified HTP ACA
Human whold blood (Table of Materials) was irradiated with different doses of 10 J/cm2 UVA to induce damage. Four different concentrations of Fpg (1, 2, 4 or 8 U/mL) were used to determine the optimal concentration for enzyme treatment in HTP ACA. The results showed that the optimal levels of DNA damage were revealed with 4 U/mL Fpg (Figure 7A). Representative comet images from UVA irradiated blood samples (Figure 7B).
Detection of DNA ICL in a representative ovarian cancer cell line using the ICL-modified HTP ACA
An ovarian cancer cell line (SKOV-3; Table of Materials) was treated with combinations of 200 µM cisplatin and/or subsequent treatment with 50 µM H2O2 for 30 min on ice. No appreciable damage was noted in the unexposed cells (Figure 8A). Exposure to H2O2 alone generated a significant MOTM (Figure 8B). In contrast, the cells in which ICL were induced showed a decreased MOTM (Figure 8C)28.
Formation and repair of cisplatin-induced DNA ICL in a representative, ovarian cancer cell line
The ICL-modified HTP ACA was used to determine the time course for DNA ICL formation and repair induced by cisplatin in an ovarian cancer cell line (A2780; Table of Materials). The cells were treated with 100 µM cisplatin for 1 h, and then incubated in cisplatin-free media (RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS)) for a subsequent time course. At various time points, the ICL-modified HTP ACA was performed to establish the levels of ICL (Figure 9)28. No ICL were detected prior to cisplatin treatment. However, after a single treatment with 100 µM cisplatin, ICL levels increased significantly, peaking at 12 h, after which levels decreased back to zero after 30 h.
Correlation between DNA ICL and DNA platinum levels
Three ovarian cancer cells were treated with 100 µM cisplatin to induce different levels of DNA-ICL, prior to analysis by the ICL-modified HTP ACA and inductively-coupled mass spectrometry (ICP-MS; see Supplementary File for details). As shown in Figure 10, differing levels of DNA-ICL were induced in the three cell lines, together with differing levels of Pt in DNA. A positive correlation (R2 = 0.9235) was observed between DNA ICL levels and platinum concentrations, indicating the association between DNA platinum levels and the corresponding ICL28.
Base excision repair in Mycoplasma-infected and uninfected BE-M17 cells
Mycoplasma infected and uninfected BE-M17 cells were treated with 50 µM H2O2 for 30 min and incubated with complete medium (Dulbecco's modified Eagle's medium supplemented with 10% (v/v) FBS) for different durations (0 min, 30 min, 1 h, 2 h, 6 h, 24 h, or 30 h) during which cells were allowed to repair. At each time point, cells were collected and frozen at -80 ˚C, in a 10% DMSO-containing medium, before performing the hOGG1-modified HTP ACA (step 6). After 30 min, levels of SB/ALS had decreased to 21% TD (percentage tail DNA) in the uninfected cells, whereas the infected cells showed 49% TD (Figure 11A). After ~15 h, levels of SB/ALS had returned to baseline in both infected and uninfected cells. For the oxidized purines, the uninfected BE-M17 initially showed a small increase in damage, before returning to baseline within 30 h (Figure 11B). In contrast, the infected cells showed a sustained, significant increase in oxidized purines, which remained elevated, and did not return to baseline levels even after 30 h (Figure 11B)23.
Figure 1: Overview of the conventional alkaline comet assay procedure. (i) A single-cell suspension of cultured cells or a sample of whole blood is mixed with 0.6% (w/v) LMP agarose. (ii) The cell/agarose mixture is applied to pre-coated microscope slides and covered with coverslip until solidified. (iii) The cells are lysed using a high pH lysis buffer overnight, forming nucleoid bodies, before (iv) washing with ddH2O. (v) The cellular DNA unwinds in the high pH electrophoresis buffer. The presence of strand breaks allows the DNA to relax and unwind, and under electrophoresis, the DNA is drawn out of the nucleoid body, forming a tail. The slides are then (vi) drained, dried, (vii) neutralized, and (viii) washed with ddH2O before (ix) being dried overnight. Slides are then (x) rehydrated with ddH2O, (xi) stained, (xii) washed, and finally (xiii) scored and analyzed, typically using fluorescent microscopy and image analysis software. This figure is reproduced from a previous publication20. Please click here to view a larger version of this figure.
Figure 2: The materials comprising the high-throughput comet electrophoresis system. HTP electrophoresis tank, HTP racks, and the dishes for lysis, wash, neutralization, and staining are shown. Please click here to view a larger version of this figure.
Figure 3: Representative images of a comet assay slide and HTP rack (microscope slide carrier). (A) For correct orientation, the pre-coated face of the microscope slide is recognized by a black dot in the right-hand corner of a microscope slide. (B) The image of the HTP rack illustrates how the slides are kept in a tight vertical orientation, with tabs on the carrier to fix its orientation within the electrophoresis tank. Each carrier can accommodate up to 25 slides. Please click here to view a larger version of this figure.
Figure 4: Representation of the chilling plate with sample slides and freezer packs in place. Please click here to view a larger version of this figure.
Figure 5: Screenshot of representative comets taken during scoring. HaCaTs (A) without treatment and (B) treated with 1 J/cm2 UVB prior to performing HTP ACA. Most software packages can calculate a variety of comet endpoints, but the most common ones are the % tail DNA (preferred) or tail moment based upon these images (blue: start of the head, green: middle of the head, and purple: end of tail). The scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 6: Representative graphs illustrating the effect of electrophoresis voltage on percentage tail DNA, determined using the HTP ACA. Cells were exposed to (A) 5 or 10 J/cm2 UVA, (B) 0.5 or 1.0 J/cm2 UVB, or (C) 50 µM H2O2 prior to the HTP ACA, with the electrophoresis voltage at either 1, 1.09, or 1.19 V/cm. Data represent the mean of 200 determinations from n = 2 duplicate experiments31. Please click here to view a larger version of this figure.
Figure 7: Representative graph and comet images of human blood analysed by the Fpg modified HTP ACA. Human blood samples were irradiated with 10 J/cm2 UVA or sham irradiated ('ctrl') on ice prior to the lysis step. Different concentrations of Fpg (1, 2, 4, or 8 U/mL) were used for the enzyme treatment prior to electrophoresis. (A) Data represent the mean ± SEM of 300 determinations from n=3 experiments. (B) Representative images of comets for each concentration of Fpg in 10 J/cm2 UVA irradiated blood samples. The scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 8: Representative comet images illustrating ICL detection following cisplatin treatment. (A) Control cells without any treatment, (B) cells which were treated with H2O2 (50 µM) only, (C) cells which were treated with H2O2 (50 µM) and cisplatin (200 µM), illustrating the tail to be shorter than in (B), due to the presence of ICL28. The scale bar is 10 µm. Please click here to view a larger version of this figure.
Figure 9: Demonstration of the kinetics of cisplatin-induced ICL formation and repair. A2780 cells were treated with 100 µM of cisplatin in the culture medium for 1 h. The cisplatin-containing medium was then removed, and the cells were cultured for various time points, before analysis by ICL-modified HTP ACA. Data represent mean ± SEM from n = 3 experiments28. **** P < 0.0001. Please click here to view a larger version of this figure.
Figure 10: Correlation between DNA ICL and platinum concentration. DNA ICL were determined by the ICL-modified HTP ACA and platinum levels were measured by ICP-MS (with Single Quad-Kinetic Energy Discrimination, SQ-KED), in three ovarian cancer cell lines. R2 = 0.9235. See Supplementary File for ICP-MS methodology to quantify platinum levels in DNA28. Please click here to view a larger version of this figure.
Figure 11: A representative graph illustrating DNA damage and repair, determined by the hOGG1-modified comet assay, in Mycoplasma infected versus uninfected BE-M17 cells. After treatment with 50 µM H2O2 for 30 min, cells were allowed to repair for different durations (0, 30 min, 1 h, 2 h, 6 h, 24 h, or 30 h). The hOGG1-modified HTP ACA was used to measure (A) SB/ALS and (B) oxidized purines in infected (red data points) and uninfected (black data points) BE-M17 cells. Data represent the mean of 200 determinations from n = 2 duplicate experiments. This figure is reproduced with permission from a previous publication23. Please click here to view a larger version of this figure.
Reagent | Stock Solution | Working solution | |
Lysis buffer | 100 mM Na2EDTA, 2.5 M NaCl, and 10 mM Tris Base in ddH2O; adjust pH to 10 with 10 M NaOH | 1% Triton X-100 in lysis stock solution | |
Electrophoresis buffer | 10 M NaOH and 200 mM Na2EDTA in ddH2O | 300 mM NaOH and 1 mM Na2EDTA; pH > 13 | |
Neutralization buffer | 0.4 M Tris Base in ddH2O; adjust pH to 7.5 with HCl | ||
Staining buffer | 1 mg/mL propidium iodide | 2.5 µg/mL propidium iodide in ddH2O |
Table 1: Composition of reagents used in HTP ACA. The stock and working concentrations of lysis, electrophoresis, neutralization, and staining buffers are shown.
Supplementary File. Please click here to download this File.
This study demonstrates the versatility provided by the current equipment, which can be used to achieve high throughput with a variety of representative, common variants of the comet assay (i.e., alkaline, enzyme-modified, blood, and ICL, and other variants will be suitable too). In addition, the present approach brings with it several benefits20,21: (a) assay run time is decreased due to manipulation of multiple slides in parallel (handling time decreases by 60%); (b) risk of damage to gels, and hence the risk to the experiment are decreased; (c) reagent requirements are decreased (e.g., the volume of the electrophoresis tank is smaller than the conventional tank); (d) the number of slides run is increased. One tank can provide a 20% increase in the number of slides run compared to a single conventional tank; however, multiple electrophoresis tanks can be run or slaved (i.e., multiple tanks controlled by a single power supply), in parallel from the same power supply, and still require a benchtop footprint smaller than a single conventional tank with ice tray; and (e) tank footprint is decreased due to vertical orientation of slides and integral cooling (saves lab space); the HTP tank comprises a high-performance ceramic cooling base with a sliding drawer that can fit one frozen cooling pack to maintain optimal buffer temperature without having to perform the process in a cold room.
Moreover, the chilling plate developed by us accommodates 26 comet slides, enables rapid solidification of the low melting point agarose on the comet assay slides and facilitates an easy retrieval of the slides after the agarose gel is solidified. The above innovations make the comet assay process simpler and easier.
While other high-throughput approaches have been developed (e.g., 12-gel comet assay, CometChip, or 96 mini-gel formats)25, many scientists prefer using the conventional microscope slides (which includes the commercially available pre-coated slides, or other specialized slides). The present approach can accommodate all types of microscope slides, allowing experiments using these slides to be scaled up through faster slide processing and handling. As noted above, the HTP comet system brings many advantages, but there is one notable limitation: the current approach provides only a 20% increase in the number of samples run, compared to a conventional horizontal tank (although processing of slides is much faster). The CometChip and 96 mini-gel formats run a greater number of samples. To date, we do not know whether the present approach can accommodate the CometChip or 96 mini-gel formats, although we predict that it will. As noted above, the number of samples can be increased further by slaving tanks to a single power supply. As with all approaches, there is still a chance of losing or damaging the gels while loading samples and analyzing them under the microscope, but this is more due to operator error, and the chances of this are minimized with the current approach.
The use of the HTP comet system can greatly help analyze DNA damage, facilitating the use of the comet assay in a wide range of applications, such as molecular epidemiology, male reproductive science, genotoxicology studies, and environmental toxicology. This is particularly true for those users who wish to have all the benefits of improved throughput, and ease of use, without moving away from the familiar, cost-effective, conventional microscope slides.
The authors have nothing to disclose.
The work reported in this publication was, in part, supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274. The content is solely the authors' responsibility and does not necessarily represent the official view of the National Institutes of Health.
22 x 22 mm glass coverslips | Fisher Scientific, Hampton, NH, USA | 631-0124 | |
A2780 | ECACC, Louis, MO, USA |
93112519 | |
Concentrated nitric acid (OptimaTM grade) | Fisher Scientific Fair Lawn, NJ, USA | A467-250 | |
Fluorescence microscope equipped with a camera | Zeiss, Jena, Germany | ||
Fresh human whole blood | Zen Bio Inc | SER-WB10ML | Commercial human whole blood sample |
GraphPad Prism | GraphPad Software, San Diego, California | Data analysis software | |
HTP Comet Assay system | Cleaver Scientific | COMPAC- 50 | |
Human Keratinocyte (HaCaTs) | American Type Culture Collection (ATCC), Manassas, VA, USA | Discontinued | Can be purchased from another company ADDEXBIO TECHNOLOGIES Cat# T0020001 |
Hydrogen peroxide (H2O2) 30% in water |
Fisher Scientific, Hampton, NH, USA | BP2633-500 | |
ICP-MS iCAP RQ ICP-MS system |
Thermo Scientific, Waltham, MA, USA |
IQLAAGGAAQFAQKMBIT | |
Image and Data Analysis software | Perceptive Instrument, Bury St Edmunds, England, UK |
125525 | Free image analysis softwared is available e.g., ImageJ |
Internal Standard Mix | SPEX Certiprep, Metuchen, NJ, USA |
CL-ISM1-500 | Bismuch (isotope monitored 209 Bi)-concnetration of 10 µg/mL in 5% HNO3 |
Low melting point Agarose | Invitrogen Waltham, MA, USA |
P4864 | |
Na2EDTA (disodium ethylenediaminetetraacetic acid) | Sigma Aldrich, St. Louis, MO, USA |
E5134 | |
NaCl (Sodium chloride) | Sigma Aldrich, St. Louis, MO, USA |
S7653 | |
NanoDrop One | Thermo Scientific, Waltham, MA, USA |
701-058108 | Nanodrop for measuring DNA concentration |
Nanopure Infinity Ultrapure Water System (Barnstead Nanopure) | Thermo Scientific, Waltham, MA, USA |
D11901 | Ultrapure water (16 MΩ cm-1) |
NaOH (sodium Hydroxide) | Sigma Aldrich, St. Louis, MO, USA |
E5134 | |
Normal melting point Agarose | Fisher Scientific, Hampton, NH, USA |
16520100 | For pre-coating slides |
OCI-P5X | University of Miami, Miami, FL, USA |
N/A | Live Tumor Culture Core facility provided the cells |
Platinum (Pt) reference standard | SPEX Certiprep, Metuchen, NJ, USA |
PLPT3-2Y | (1000 µg/mL in 10% HCl) containing Bismuch |
Propidium Iodide (1.0 mg/mL in water) |
Sigma Aldrich, St. Louis, MO, USA |
12-541BP486410ML | |
QIAamp DNA Mini Kit | Qiagen Valencia, CA, USA |
51304 | DNA extraction Kit |
Single-frosted glass microscope slides | Fisher Scientific, Hampton, NH, USA |
12-541B | |
SKOV3 | ECACC, Louis, MO, USA |
91091004 | |
Slide box | Fisher Scientific, Hampton, NH, USA |
03-448-2 | Light proof, to protect cells from the formation adventitious damage (according to the widely held view) and prevent fading of the fluorescent dye |
Slide Chilling plate | Cleaver Scientific, Rugby, England, UK |
CSL-CHILLPLATE | |
Treatment dish | Cleaver Scientific, Rugby, England, UK |
STAINDISH4X | |
Tris-base | Sigma Aldrich, St. Louis, MO, USA |
93362 | |
Triton X-100 | Fisher Scientific, Hampton, NH, USA |
BP151-500 | |
Trypsin EDTA (0.5%) | Invitrogen Gibco, Waltham, MA, USA |
15400054 | |
Vertical Slide Carrier | Cleaver Scientific, Rugby, England, UK |
COMPAC-25 |