We thank the Dungrawala lab members for their input and feedback. This work was funded by NIH grant R35GM137800 to HD.

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
<ol>
	<li>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.</li>
	<li>Once melted, quickly aliquot 100 &#181;L of the agarose to 1.5 mL centrifuge tubes as required and store the tubes at 4 &#176;C. The solidified agarose can be stored at 4 &#176;C for up to 1 year.</li>
	<li>On the day of the experiment, remove the required number of centrifuge tubes from the 4 &#176;C storage. Melt the agarose by placing the tubes in a heat block or water bath at 42 &#176;C. 
	NOTE: Place the tubes at 42 &#176;C prior to harvesting the cells to ensure the agarose is completely melted for resuspension (step 3.7).</li>
	<li>Prepare fresh lysis buffer and TAE buffer on the day of the experiment and store them at room temperature and 4 &#176;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).</li>
	<li>Mark the sample identifications on the frosted end of the 2-well comet slides and store them at room temperature until ready to use.</li>
</ol>
2. Preparation of samples
<ol>
	<li>Prior to the day of the experiment, seed the cells in a 6-well dish and culture overnight at 37 &#176;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.</li>
	<li>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 &#181;M CPT for 1 h. CPT is a topoisomerase-1 inhibitor that induces double-strand breaks in a replication-dependent manner14.</li>
</ol>
3. Cell resuspension and lysis
<ol>
	<li>Aspirate cell culture media and rinse twice with 1x PBS.</li>
	<li>Add 300 &#181;L 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.</li>
	<li>Add 700 &#181;L 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.</li>
	<li>Centrifuge the cell suspension at 2400 x g&#160;for 5 min at room temperature.</li>
	<li>Aspirate the media and resuspend the cells in 1 mL of cold 1x PBS.</li>
	<li>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.</li>
	<li>Resuspend 10 &#181;L of the cell suspension in 100 &#181;L 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 &#176;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)</li>
	<li>Store the slides at 4 &#176;C in the dark for 15-20 min to ensure the agarose has completely solidified (Figure 2A).</li>
	<li>Place the slides in Coplin jars and immerse the slides in lysis buffer for 1 h at room temperature.</li>
	<li>Rinse the slides once with cold TAE buffer and immerse the slides in cold TAE buffer for 30 min at 4 &#176;C.</li>
</ol>
4. Electrophoresis
<ol>
	<li>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.</li>
	<li>Pour approximately ~850 mL of cold 1x TAE buffer into the apparatus.</li>
	<li>Remove the slides from 4 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
5. Staining
<ol>
	<li>After completion of electrophoresis, remove the slides from the apparatus and immerse them in DNA precipitation solution for 30 min at room temperature.</li>
	<li>Discard the DNA precipitation solution and immerse the slides in 70% ethanol solution for 30 minu at room temperature.</li>
	<li>Discard the 70% ethanol solution. Remove the slides and dry them in a hybridization oven at 45 &#176;C for 30 min in the dark (Figure 2B). 
	NOTE: Slides can be left in a dark, dry place overnight for the drying step.</li>
	<li>Incubate the wells with 100 &#181;L of 1x SYBR Green solution for 30 min at room temperature in the dark.</li>
	<li>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.</li>
	<li>Place the slides in a drawer and allow to dry for 30 min.</li>
	<li>Proceed with imaging of the slides.</li>
</ol>
6. Imaging and analyses
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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&#160;Figure 5.</li>
	<li>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&#39;s multiple comparisons if comparing three or more samples.</li>
</ol>

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+A+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: A method to measure DNA damage in individual cells.</a> Nat Protoc. 1 (1), 23-29 (2006).</li><li>De Lapuente, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+and+its+applications+in+the+field+of+ecotoxicology:+A+mature+tool+that+continues+to+expand+its+perspectives.">The comet assay and its applications in the field of ecotoxicology: A mature tool that continues to expand its perspectives.</a> Front Genet. 6, 180 (2015).</li><li>Azqueta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+comet+assay+in+human+biomonitoring:+An+hcomet+perspective.">Application of the comet assay in human biomonitoring: An hcomet perspective.</a> Mutat Res Rev Mutat Res. 783, 108288 (2020).</li><li>Speit, G., Hartmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+A+sensitive+genotoxicity+test+for+the+detection+of+DNA+damage.">The comet assay: A sensitive genotoxicity test for the detection of DNA damage.</a> Methods Mol Biol. 291, 85-95 (2005).</li><li>Collins, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+DNA+modifications+with+the+comet+assay:+A+compendium+of+protocols.">Measuring DNA modifications with the comet assay: A compendium of protocols.</a> Nat Protoc. 18 (3), 929-989 (2023).</li><li>Rapp, A., Hausmann, M., Greulich, K. 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The authors declare no competing interests.

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 r...

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 immunofluorescence1,2. 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 stressors10,11. 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 inhibitors16,17. 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 phenotypes18,19,20,21. 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 proteins22,23, analysis of replication intermediates24,25, and investigation of transcription-replication conflicts in genome maintenance26,27. 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.

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

detection of dna breaks in dividing human cells by neutral comet assay

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.

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...

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

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.

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

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...

detection-of-dna-breaks-in-dividing-human-cells-by-neutral-comet-assay

Research

JoVE Journal

Bioengineering

373 Views.  University of South Florida, Tampa. 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.

Video: Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Cancer Research

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Genetics

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Neuroscience

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

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.

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 ...

Biology

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app.jove.com/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Test komety do wykrywania uszkodzeń DNA w komórkach

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...

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

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Rozdziały w tym wideo

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

Evaluating In Vitro DNA Damage Using Comet Assay

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Test komety do wykrywania uszkodzeń DNA w komórkach