Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

Summary

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

Abstract

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

Introduction

The comet assay is a gel electrophoresis method used to detect DNA breaks at the single-cell level. It relies on the principle that open-ended DNA breaks migrate under electrophoretic conditions, while intact DNA remains largely static. Broken DNA migrates because breaks result in the relaxation of supercoiled DNA, causing its gradual spatial relocation towards the anode in the electrophoretic chamber, which results in a comet-like appearance observed by immunofluorescence^1,2. The extent of DNA damage is then measured by quantifying the amount of broken DNA that has migrated relative to the compact DNA.

The two most commonly used comet assay methods are the alkaline comet (AC) assay and the neutral comet (NC) assay. The AC assay is performed under denaturing conditions using a high alkaline pH solution, while the NC assay is carried out in a solution with neutral pH. Both NC and AC assays can reliably detect DNA breaks that occur in the nucleus. However, the alkaline version is also advantageous for detecting alkali-labile sites³. Both formats of the assay require the use of lysis conditions to ensure that DNA is free of proteins prior to electrophoresis.

This assay is a simple method for detecting DNA breaks and offers several unique advantages for determining cellular DNA damage. The method is relatively easy to set up and requires reagents that can either be prepared in the laboratory or purchased from commercial vendors. As a single-cell resolution technique, the assay requires very little starting material. Notably, the NC assay can measure breaks with high sensitivity, reported to detect between 50 and 10,000 breaks per cell⁴. The versatility of this technique is demonstrated by its wide range of applications, including ecotoxicology⁵, human biomonitoring⁶, and genotoxicity studies⁷. The assay can also be reliably used across various cell types and adapted for high-throughput assays⁸ and for assessing breaks at specific genomic regions⁹. Thus, this assay serves as a rapid and reliable technique for investigating break formation at the single-cell level.

The process of DNA replication is constantly challenged by several endogenous and exogenous stressors^10,11. Stalling of replisomes due to such lesions can cause DNA breaks and result in heightened genome instability. DNA breaks also often arise as replication intermediates to facilitate DNA repair¹². Additionally, several chemotherapeutic agents induce replication-dependent double-strand breaks (DSBs), such as camptothecin (CPT)^13,14,15and PARP inhibitors^16,17. Thus, this assay serves as a powerful methodology to study the nature of DNA lesions, assess the processing of replication forks, and investigate the therapeutic potential of DNA-damaging agents. Adaptations of the assay that involve labeling newly synthesized DNA with BrdU have been useful for studying replication-associated stress phenotypes^18,19,20,21. Notably, the NC assay is a reliable method for studying DNA break induction in the context of replication, as demonstrated in studies involving the characterization of fork proteins^22,23, analysis of replication intermediates^24,25, and investigation of transcription-replication conflicts in genome maintenance^26,27. Herein, we outline a NC assay protocol with the goal of quantifying DNA breaks during replication in human cells. This protocol can be readily implemented by researchers interested in assessing replication stress-associated damage in a wide variety of dividing human cells.

Protocol

This protocol (Figure 1) primarily utilizes adherent U2OS cancer cell lines but is adaptable to a wide variety of adherent and suspension cells grown in tissue culture. The solutions and buffers used in this study are detailed in Table 1. The reagents and equipment used are listed in the Table of Materials.

1. Preparation of materials

1. Prior to the day of the experiment, place the provided bottle with low melting agarose (1% low melting agarose in PBS) in a hot water bath for 5-10 min to ensure the agarose has completely melted.
   NOTE: Do not heat the bottle in the microwave.
2. Once melted, quickly aliquot 100 µL of the agarose to 1.5 mL centrifuge tubes as required and store the tubes at 4 °C. The solidified agarose can be stored at 4 °C for up to 1 year.
3. On the day of the experiment, remove the required number of centrifuge tubes from the 4 °C storage. Melt the agarose by placing the tubes in a heat block or water bath at 42 °C.
   NOTE: Place the tubes at 42 °C prior to harvesting the cells to ensure the agarose is completely melted for resuspension (step 3.7).
4. Prepare fresh lysis buffer and TAE buffer on the day of the experiment and store them at room temperature and 4 °C, respectively, until ready to use. In addition, aliquot 1x PBS in 15 mL conical tube and place it in the fridge to use in the resuspension step (step 3.5).
5. Mark the sample identifications on the frosted end of the 2-well comet slides and store them at room temperature until ready to use.

2. Preparation of samples

1. Prior to the day of the experiment, seed the cells in a 6-well dish and culture overnight at 37 °C with 5% CO₂. For U2OS cells, approximately 2-3 x 10⁵ cells were seeded for each sample.
   NOTE: Cell numbers might need to be adjusted depending on the cell type used for analyses.
2. On the day of the experiment, treat cells with the conditions as per the experimental design. As a positive control to demonstrate replication-dependent break induction, treat cells with 1 µM CPT for 1 h. CPT is a topoisomerase-1 inhibitor that induces double-strand breaks in a replication-dependent manner¹⁴.

3. Cell resuspension and lysis

1. Aspirate cell culture media and rinse twice with 1x PBS.
2. Add 300 µL of trypsin to each well. Incubate the dish in a tissue culture incubator for 2-3 min.
   NOTE: Prolonged incubation in trypsin can generate spurious results during comet analyses.
3. Add 700 µL of cell culture media to each well. Resuspend gently and transfer the samples to 1.5 mL centrifuge tubes.
   NOTE: Counting cells at this step is recommended to obtain the appropriate cell density in step 3.6 to reduce the frequency of overlapping tails during analysis.
4. Centrifuge the cell suspension at 2400 x g for 5 min at room temperature.
5. Aspirate the media and resuspend the cells in 1 mL of cold 1x PBS.
6. Repeat step 3.4 and step 3.5 twice and resuspend the cells in cold 1x PBS at a cell density of 2 x 10⁵ cells per ml.
   NOTE: Assessing cell viability at this step is recommended to ensure an adequate number of viable cells are present. At least 90% viability is recommended.
7. Resuspend 10 µL of the cell suspension in 100 µL aliquot of melted agarose, and using the same pipette tip, mix gently and spread the mixture evenly into one well of the slide. Repeat this step for all samples as needed.
   NOTE: Avoid removing all aliquots from 42 °C water bath at once to prevent the agarose from solidifying. This step should preferably be performed by the water bath, and each sample is resuspended and spread in a staggered manner. Avoid introducing bubbles while spreading the agarose on the slide since the air pockets will affect the migration of the comet tails during electrophoresis (step 4.5) and introduce cracks in the drying step (step 5.3)
8. Store the slides at 4 °C in the dark for 15-20 min to ensure the agarose has completely solidified (Figure 2A).
9. Place the slides in Coplin jars and immerse the slides in lysis buffer for 1 h at room temperature.
10. Rinse the slides once with cold TAE buffer and immerse the slides in cold TAE buffer for 30 min at 4 °C.

4. Electrophoresis

1. Setup the electrophoretic chamber on a flat surface and level the system using the four leveling knobs on the edges of the apparatus. Insert cold packs in the bottom chamber to keep the solution cold during electrophoresis.
2. Pour approximately ~850 mL of cold 1x TAE buffer into the apparatus.
3. Remove the slides from 4 °C and place them on the electrophoresis tray in the proper orientation to allow for the migration of broken DNA.
   NOTE: The tray holds ten 2-well slides. Place the slides in the center of the tray if working with less number of samples. If working with an odd number of slides, include a blank slide such that each well accommodates 2 slides.
4. Gently place the provided slide tray overlay on top of the tray to cover the slides.
   NOTE: For proper operation of the apparatus, the volume of the TAE buffer should not be lower than the bottom surface of the overlay and not exceed the top surface of the overlay.
5. After connecting the cables, set the power at 21 V and perform electrophoresis (1 V/cm) for 40 min.
   NOTE: The electrophoresis time may need to be adjusted depending on the cell type used for analyses. Long tails in untreated samples indicate excessive runtime. Conversely, short tails in positive control samples may reflect insufficient runtime.

5. Staining

1. After completion of electrophoresis, remove the slides from the apparatus and immerse them in DNA precipitation solution for 30 min at room temperature.
2. Discard the DNA precipitation solution and immerse the slides in 70% ethanol solution for 30 minu at room temperature.
3. Discard the 70% ethanol solution. Remove the slides and dry them in a hybridization oven at 45 °C for 30 min in the dark (Figure 2B).
   NOTE: Slides can be left in a dark, dry place overnight for the drying step.
4. Incubate the wells with 100 µL of 1x SYBR Green solution for 30 min at room temperature in the dark.
5. Decant the SYBR Green solution by tilting the slides and removing the excess by gently dabbing the edge of the well with a lint-free wipe.
6. Place the slides in a drawer and allow to dry for 30 min.
7. Proceed with imaging of the slides.

6. Imaging and analyses

1. Setup the microscope for imaging purposes.
   NOTE: All images were acquired using a epifluorescence microscope. 10x magnification with 0.45 numerical aperture was used with an exposure time of 1/40s and a gain of +6dB. The capturing mode of monochrome 8bit was utilized with 2 x 2 binning and black balance correction.
2. Place the slides on the microscope stage and begin imaging using the FITC filter.
   NOTE: Use the untreated sample first to test exposure time. Most cells do not exhibit comet tails since DNA is more compact. Switch to the well with CPT-treated cells and image using the same exposure time. Comet tails with increased lengths will be observed in this sample due to the migration of broken DNA ends.
3. Acquire images for at least 100 comets per sample using the same exposure time across three technical replicates.
   NOTE: Comet tails at the end of coverslips tend to migrate unevenly and act as outliers in the sample distribution. Most of the comets captured for analyses are present close to the center of the coverslip (Figure 3).
4. Export images and score the comets either manually (using Image J) or by using freely available comet scoring software. Comets that are individually distinguishable and fully contained in the field of view are selected for scoring. Overlapping or partially visible comets are not considered for further analyses (Figure 4).
   NOTE: Users can score comets on Image J using the following plugin: https://cometbio.org/index.html. Details for scoring comets using CometScore software are included in Figure 5.
5. Plot tail moments acquired from the analyses as box and whisker plots depicting 10-90 percentile values. Perform normality tests to determine whether parametric, or non-parametric tests should be utilized for statistical analyses.
   NOTE: In most cases, non-parametric tests are applied using the Mann-Whitney test if comparing two samples only or the Kruskal-Wallis test with Dunn's multiple comparisons if comparing three or more samples.

Results

Analysis of break induction by replication stress reagents
U2OS cells were treated with DMSO, 4 of mM hydroxyurea (HU), or 100 nM of CPT for 4 h and analyzed for break accumulation by the NC assay (Figure 6A). While CPT induces breaks during S-phase by blocking topoisomerase-1¹³, HU-induced depletion of nucleotide pools stalls replication forks that are progressively converted into DSBs²⁸. The data indicates that exposure to either HU or CPT triggers break induction in S-phase, which is detectable by NC assay.

Analysis of break induction comparing lysis at different temperatures
Untreated U2OS cells were incubated in lysis buffer either at room temperature (RT) or 4 °C prior to analyses of break induction (Figure 6B). The results indicate that the temperature at which lysis is performed has minimal impact on the migration of tails in the assay.

Temporal analysis of breaks by hydroxyurea (HU)
U2OS cells were left either untreated or treated with 4 mM of HU for 4 h and 8 h prior to analyses of break induction by the NC assay (Figure 7). The results indicate that prolonged stalling of replication forks with HU causes replication fork collapse into DSBs.

Dose-dependent analysis of breaks by camptothecin
U2OS cells were treated with increased doses of CPT for 1 h and harvested for break analyses using the NC assay (Figure 8). Whereas break accumulation is minimal in cells not treated with CPT, the degree of break induction gradually increases with increased dosage of CPT, thus demonstrating the utility of the assay in studying dose-dependent effects on break accumulation during DNA replication.

Comparative analysis of breaks across multiple cell types
Basal levels of DNA breaks were assessed across three divergent mitotic cell types- hTERT immortalized retinal pigment epithelial cells (hTERT-RPE-1), simian virus 40 (SV40) large T-antigen expressing human embryonic kidney cells (HEK293T) and bone osteosarcoma cells (U2OS) (Figure 9). Each cell line demonstrated differential migration of tail moments in the absence of added replication stress, which may be due to differences in cell cycle distribution, chromatin organization, or number of active replication forks. Each cell type was also exposed to 1 µM of CPT for 1 h to validate an increase in replication-dependent DNA breaks.

Analyses of break induction by AC assay
U2OS cells were treated with DMSO, 4 mM of HU, 1 µM of CPT, or 10 µM of PARP inhibitor (PARPi) Olaparib and analyzed for break induction (Figure 10). Cells were treated with DMSO, HU, and PARPi for 2 h while CPT treatment was for 1 h. The results indicate that exposure to replication stress causes break accumulation, which is observable by the AC assay.

Figure 1: Schematic representation of the NC assay. (A) The required number of cells is seeded the day before in a 6-well dish. (B) After drug treatments, the cells are trypsinized and harvested in cold PBS. (C) The cells are gently resuspended in melted agarose at a ratio of 10:1 (10 µL of agarose for 1 µL of cell suspension) using a pipette tip. (D) The cell suspension is transferred onto a slide well using a pipette tip and spread uniformly to cover the well surface completely with agarose. (E) Following incubation at 4 °C, the slides are immersed in lysis buffer, followed by cold 1x TAE buffer. (F) The samples are subjected to electrophoresis using the appropriate apparatus. (G) The slides are then immersed in the DNA precipitation solution, followed by a 70% ethanol solution. (H) After drying, the wells are stained with 1x SYBR Green solution. (I) The slides are imaged using an epifluorescence microscope. Please click here to view a larger version of this figure.

Figure 2: Side view of agarose on comet slide wells. (A) After incubation at 4 °C (step 3.8), the solidified agarose appears as a 'bubble' with a convex shape. (B) After drying at 45 °C (step 5.3), the agarose appears flattened on the well. Please click here to view a larger version of this figure.

Figure 3: Imaging and analysis of comets. (A) Comets within the inner circle of the well (highlighted in blue) are selected for analysis. Comets toward the outer edge of the well are excluded due to atypically longer tail moments, marking them as outliers. (B) At least 100 representative comets are randomly selected from the blue-colored region. Please click here to view a larger version of this figure.

Figure 4: Representative image of comets obtained from the NC assay. The right panel displays a representative field of view for SYBR Green-stained nuclei, while the left panel highlights the region (green outline) used for scoring comets. Nuclei that are partially visible or stacked close to each other (red outline, right panel) are not included in the analysis. Comets selected for scoring should be individually distinguishable, fully contained within the field of view, and exhibit uniform staining intensity. Please click here to view a larger version of this figure.

Figure 5: Representative scoring of comets obtained from the NC assay. (A) Individual nuclei are outlined using CometScore software. (B) The full spectrum view (left panel) is enabled, and the threshold (cutoff bar) is adjusted to achieve optimal signal intensity with minimal background. The 'full spectrum' signal should correspond to the 'single hue' image (top image, right panel) and not exceed the outline (bottom image, right panel). (C) The vertical yellow line in the box is aligned with the center of the comet head, using the widest part of the comet head as a reference for consistency. (D) The score for each analyzed comet is reported in the upper right corner. Please click here to view a larger version of this figure.

Figure 6: Analysis of break induction by replication stress reagents. (A) U2OS cells were treated with DMSO, 100 nM camptothecin (CPT), or 4 mM hydroxyurea (HU) before break induction analysis. The representative graph shows increased break accumulation in cells treated with CPT or HU. (B) Untreated U2OS cells were seeded on comet slides and incubated for lysis (step 3.9) either at room temperature (RT) or 4 °C to compare the effect of lysis temperature. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.

Figure 7: Temporal analysis of breaks induced by hydroxyurea. U2OS cells were either untreated or treated with 4 mM HU for 4 h or 8 h before break induction analysis. The representative graph depicts a gradual increase in break accumulation in cells treated with HU. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.

Figure 8: Dose-dependent analysis of breaks induced by camptothecin. U2OS cells were treated with either DMSO or increasing concentrations of CPT (25 nM, 100 nM, 500 nM, and 1 µM) for 1 h before break analysis. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. The representative graph shows a dose-dependent increase in breaks induced by CPT. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.

Figure 9: Comparative analysis of breaks across multiple cell types. hTERT-RPE-1, HEK293T, and U2OS cells were treated with either DMSO or 1 µM CPT for 1 h. The representative graph shows the relative extent of DNA damage across the three cell types in the presence or absence of CPT. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.

Figure 10: Analysis of break induction by the AC assay. U2OS cells were treated with DMSO, 4 mM HU, 1 µM CPT, or 10 µM PARP inhibitor Olaparib prior to break induction analysis. Cells were treated with DMSO, HU, and PARP inhibitor for 2 h, while CPT treatment was for 1 h. The representative graph demonstrates increased break accumulation in cells treated with CPT, HU, or PARP inhibitors. Tail moments from the analyses are plotted as box-and-whisker plots depicting 10-90 percentile values, with median values represented by horizontal lines within the boxes. P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparisons (cutoff p-value of 0.05). Please click here to view a larger version of this figure.

Reagent	Working solution
Lysis Buffer	0.5% SDS, 200 mM Tris-Cl (pH 7.4), 50 mM EDTA
1x TAE Buffer	40 mM Tris Base, 20 mM Acetic Acid, 1 mM EDTA in pH 8.45
DNA Precipitation solution	1 M Ammonium Acetate, 87% Ethanol
70% Ethanol solution	70% Ethanol in distilled water
1x SYBR Green solution	1x SYBR Green in 1x TAE buffer
DMEM culture media	DMEM with 7.5% FBS
DMEM/F12 culture media	DMEM/F12 with 7.5% FBS
Alkaline unwinding solution (for use in Alkaline Comet Assay)	200 mM NaOH, 1 mM EDTA in pH>13

Table 1: List of buffers and solutions.

Discussion

This protocol outlines a NC assay for assessing DNA breaks in human cells undergoing active replication. The protocol is relatively simple to perform, and researchers can readily adapt it to high pH conditions for alkaline analyses⁸. It includes two critical steps, as described in steps 3.7 and 4.5. In step 3.7, it is essential to spread the melted agarose with cells uniformly across the well to prevent comets from overlapping during analysis. Ensure that cellular aggregates are dissolved before resuspending the cells in agarose. Preventing the release of the pipette tip while spreading the agarose will help minimize the introduction of bubbles. The conditions for electrophoresis are crucial for achieving appropriate comet tail migration for imaging and analysis. Researchers should consider the nature of the cell lines used for analysis, as the extent of tail migration and the percentage of cells exhibiting comet tails vary across cell types (Figure 9). Therefore, it is recommended to empirically determine the electrophoresis run time prior to experimentation.

If no comet tails are observed in positive control samples (CPT-treated cells), the drug efficacy should be corroborated in parallel using alternative approaches, such as immunostaining for the DNA damage marker γH2AX. Extensive tail migration can preclude the analysis of break induction. Minimizing electrophoresis time and assessing cell morphology prior to analysis can prevent obtaining comet tails that are too long. Insufficient drying of the slides can result in blurry and hazy comet images. Ensure that the agarose on the comet slide is evenly dried and flattened before staining with 1x SYBR solution. If the range of fold induction is narrow, performing a dose-response curve with CPT can help determine an optimal working range for experimental purposes. Applying these troubleshooting tips will help maximize the results from these assays.

This assay methodology is cost-effective and highly sensitive compared to other techniques used to study DNA break induction, such as the Halo assay^29,30, TUNEL assay³¹, and pulsed-field gel electrophoresis³². However, the current assay technique has certain limitations. It does not provide a precise number of DNA lesions but offers a relative estimation of break accumulation compared to undamaged cells. Additionally, it cannot assess break induction that occurs in a cell cycle-dependent manner. To exclude changes in break induction due to differences in cell cycle progression, cell cycle analyses by flow cytometry should be performed in parallel.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x DPBS	Gibco	14190144
Camptothecin	Selleckchem	S1288
Comet LMAgarose (LMA)	R&D systems	4250-050-02
CometAssay Electrophoresis System II	R&D systems	4250-050-ES	Includes electrophoresis tank, safety lid, cables, 2/20 wells slide trays and slide tray overlay
CometScore software	TriTek		open-sourced
CometSlide	R&D systems	4250-050-03
DMEM, high glucose	Gibco	11965092
DMEM/F12	Gibco	11320033
DMSO	FisherSci	D128-4
Epifluorescence microscope	Keyence	BZ-X810
Fetal Bovine Serum - Premium	Bio-Techne	S11150
Gibco Trypsin-EDTA (0.05%), phenol red	Gibco	25300054
GraphPad Prism 10.0	GraphPad
HEK293T cells	ATCC	CRL-11268
hTERT-RPE-1 cells	ATCC	CRL-4000
Hydroxyurea	Millipore Sigma	H8627
PARP inhibitor Olaparib	Selleckchem	S8096
PowerPac	FisherSci	FB300Q
Surface Treated Sterile Tissue Culture Plate	FisherSci	FB012927
SYBR Green I Nucleic Acid Gel Stain (10,000x)	FisherSci	S7567
U2OS cells	ATCC	HTB-96
UVP HB-1000 Hybridization Incubator	FisherSci	UVP95003001