This work was supported by the IMPACT Award from the Department of Radiation Oncology, University of Alabama-Birmingham Comprehensive Cancer Center, the Fighting Children's Cancer Foundation, and the Gabrielle's Angel Foundation (to ESY.).

1. Cell Culture
 <ol>
 <li>Culture neuronal cells and treat them as needed.</li>
 <li>Harvest cells into 15 ml tubes: aspirate media, rinse with phosphate buffered saline (PBS, calcium and magnesium free), add trypsin, collect in 15 ml tubes and neutralize trypsin with appropriate serum containing media.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media.</li>
 <li>Resuspend cells in PBS.</li>
 <li>Spin at 1,000 x g for 5 min.</li>
 <li>Aspirate media and resuspend cells in fresh PBS.</li>
 <li>Count cells using a hemacytometer or your preferred cell counter.</li>
 <li>Cell samples should be prepared immediately before starting the assay.</li>
 <li>All samples should be handled in the dark or yellow light to prevent DNA damage from ultraviolet light.</li>
 </ol>
 2. Comet Assay
 1. Slide Preparation
 <ol>
 <li>Melt 1% agarose (1 g / 100 ml in 1X Tris Base, Boric acid, EDTA, TBE) in a microwave for 3 min until all granules disappear.</li>
 <li>Dip slides into the molten agarose and wipe a side clean with a kimwipe. Allow the agarose to air-dry to a transparent film. This can be done in advance and slides can be stored.</li>
 </ol>
 2. Neutral Comet Assay
 <ol>
 <li>Chill lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100, pH 10) at 4 °C for at least 1 hr before use.</li>
 <li>2.2.2. Melt 1% low melting point agarose (1 g / 100ml in 1X Tris Base, Boric acid, EDTA) in a microwave for 3 min until all granules disappear. The agarose needs to be cooled to 37 °C in a water bath to avoid artificial induction of comet tail. Ideally, the agarose needs to be cooled for half an hour before use.</li>
 <li>2.2.3. Dilute the cell suspension so that there are 100,000 cells per ml. Combine the cell suspension with the low melting point agarose (at 37 °C) at a ratio of 1:10 (v/v), vortex briefly, and immediately pipette 50 μl onto the Comet Slide. Use the side of the pipette tip to spread the cell suspension evenly over the sample area. Each treatment group should be at least in triplicate.</li>
 <li>Place slides flat in refrigerator for 30 min until a circle appears in the periphery of the slide.</li>
 <li>Prechill the lysis solution and submerge slides in this solution for 30 min in the dark at 4 °C (or in a refrigerator).</li>
 <li>Pour off or aspirate the lysis solution and add 1X neutral electrophoresis buffer (Tris base, Boric acid, EDTA, 1X TBE). Leave slides in this buffer for half an hour in the refrigerator.</li>
 <li>Add prechilled 1X Neutral Electrophoresis Buffer (TBE) in electrophoresis chamber, place slides in electrophoresis slide tray. Align slides so that they are equidistant from electrodes.</li>
 <li>Pour 1X neutral electrophoresis buffer up to 0.2 inches above slides. Excess buffer will interfere with electrophoresis.</li>
 <li>Set power supply voltage to 1 V per cm (measured electrode to electrode) and run for 30 min at 4 °C (or the cold room) in the dark.</li>
 </ol>
 3. Alkaline Comet Assay
 <ol>
 <li>Chill lysis solution (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100, pH 10) at 4 °C for at least 1 hr before use.</li>
 <li>Melt 1% low melting point agarose (1 g / 100 ml in 1X Tris Base, Boric acid, EDTA) in a microwave for 3 min until all granules disappear. Then cool in a 37 °C water bath for at least 30 min.</li>
 <li>Combine cells at 1 x 105 / ml with molten low melting point agarose (at 37 °C) at a ratio of 1:10 (v/v), vortex briefly, and immediately pipette 50 μl onto the Comet Slide. Use the side of the pipette tip to spread the cell suspension evenly over the sample area. Each treatment group should be at least in triplicate.</li>
 <li>Place slides flat in refrigerator for 30 min until a circle appears in the periphery of the slide.</li>
 <li>Immerse slides in prechilled lysis solution and leave at 4 °C for 1 hr to overnight in the dark.</li>
 <li>Remove the slides from the lysis solution, drain the slides and rinse once with cold neutralization buffer for 5 min to remove residual detergent and salts prior to the alkali-unwinding step.</li>
 <li>Place slides in a gel electrophoresis chamber filled with prechilled freshly made electrophoresis Buffer (300 mM NaOH, 1 mM EDTA, pH&gt;13) not to exceed 0.5 cm above slides. Align slides so that they are equidistant from electrodes.</li>
 <li>Let slides sit in the alkaline buffer for 30 min in the dark to allow for unwinding of the DNA and the expression of alkali-liable damage.</li>
 <li>Set power supply voltage to 1 V per cm (measured electrode to electrode) and run for 30 min at 4 °C (or the cold room).</li>
</ol>
 4. Fixing and Staining Cells
 <ol>
 <li>Drain excess Electrophoresis Buffer</li>
 <li>Immerse slides in pre-chilled distilled water for 5 min at RT.</li>
 <li>Immerse slides in pre-chilled 70% ethanol for 5 min at RT.</li>
 <li>Dry samples overnight. Do not expose slides to bright light. Samples may be stored for months at room temperature prior to scoring at this stage.</li>
 <li>Stain slides by immersing in Sybr green (1X diluted in PBS) for 20 min in the refrigerator.</li>
 <li>Remove slides and allow them to dry completely in the dark. The agarose will become transparent when completely dry.</li>
</ol>
4. Image Acquisition and Analysis
 <ol>
 <li>Acquire images using fluorescent microscope set to the green filter (Zeiss AxioVision) and analyze using Comet Assay software (Perceptive Instruments).</li>
 <li>Click on the "comet head" (the nucleus) and the software calculates the parameters including the mean tail moment and amount of DNA in the nucleus.</li>
 <li>Analyze at least 200 cells per treatment.</li>
 <li>Export data to Microsoft Excel.</li>
 <li>Calculate mean tail moment for each treatment group and plot data as appropriate.</li>
 </ol>
 5. Representative Results
 An example of comet assay analysis on neuronal cells is shown in Figure 2 and Figure 3. In this case, irradiation of the neuronal cells induces DNA damage. As the cells are subjected to the electrical field, the DNA migrates at different rates due to differences in size which is subsequently analyzed using the Comet Assay software. The more the DNA damage, the farther the DNA migrates out of the nucleus. This decreases the fluorescent intensity in the nucleus which is subsequently picked up by the software and results in higher tail moment. Table 1 depicts a representative table from comet assay analysis and Figure 4 shows a representative graph comparing DNA damage in neuronal cells following radiation as measured by the comet assay.
 <img src="/files/ftp_upload/50049/50049fig1.jpg" alt="Comet assay procedure; diagram illustrating cell preparation, electrophoresis, and fluorescence analysis." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Flow chart of the comet assay. <a href="https://www.jove.com/files/ftp_upload/50049/50049fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50049/50049fig2.jpg" alt="Comet assay diagram, hippocampal neuron DNA damage analysis, fluorescent microscopy results." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Representative images of neuronal cells (A) without and (B) with comet tail.
 <img src="/files/ftp_upload/50049/50049fig3.jpg" alt="Microscope image analysis; luminous intensity distribution; particle detection; fluorescence experiment." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Comet assay analysis using Comet Assay software. (A) Representative screen shots of image acquired using Carl Zeiss fluorescent microscope and analyzed using Comet Assay software. (B) Clicking on the nucleus or the "comet head" (circled in green) generates a fluorescent map and graph (circled in yellow) and a (C) data table (circled in red). <a href="https://www.jove.com/files/ftp_upload/50049/50049fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
 <img src="/files/ftp_upload/50049/50049fig4.jpg" alt="Bar chart comparing mean tail moment in no IR vs. IR conditions; statistical analysis result." data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Representative graph obtained by plotting the mean tail moment obtained by analyzing DNA damage in irradiated and non-irradiated neuronal cells using neutral comet assay. As expected, 3Gy radiation (x-ray) induces DNA damage as depicted by the higher mean tail moment in the irradiated neuronal cells. Shown is the mean tail moment (+/- Standard error), **P&lt;0.01.
 <img src="/files/ftp_upload/50049/50049table1.jpg" alt="Data table showing head and tail metrics including length, intensity, and grey level for analysis." data-altupdatedat="1747911167000" data-alt="Table 1"> 
Table 1. Representative table obtained by comet assay analysis on irradiated and non-irradiated neuronal cells. <a href="https://www.jove.com/files/ftp_upload/50049/50049table1large.jpg" target="_blank"> Click here to view larger figure</a>.

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No conflicts of interest declared.

The comet assay has the unique capacity of analyzing individual cells. This is advantageous in identification of subpopulations of cells that demonstrate differential response to cytotoxic agents. A few practical limitations have to be taken into account. The number of cells that can be evaluated individually may vary depending on the individual. The sample size needs to be increased if there is variance in DNA damage within a population. Viable single-cell suspension is critical for this assay since predominant presence...

<table><tbody><tr><td>1.5 ml tubes</td><td>Santa Cruz Biotechnology, Inc</td><td>Sc-200271</td><td></td></tr><tr><td>10X TBE (Tris base, boric acid, EDTA)</td><td>Fisher Scientific</td><td>BP13331</td><td></td></tr><tr><td>15 ml tube</td><td>Fisher Scientific</td><td>0553851</td><td></td></tr><tr><td>Agarose</td><td>Sigma</td><td>A5093</td><td></td></tr><tr><td>Aluminum foil</td><td>Fisher Scientific</td><td>01213101</td><td></td></tr><tr><td>Beaker</td><td>Fisher Scientific</td><td>FB102300</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific</td><td>75004261PR</td><td></td></tr><tr><td>Comet Assay software</td><td>Comet Assay IV Image Analysis System</td><td></td><td></td></tr><tr><td>Cylinder</td><td>Fisher Scientific</td><td>08555F</td><td></td></tr><tr><td>EDTA</td><td>GIBCO</td><td>15575</td><td></td></tr><tr><td>Electrophoresis chamber</td><td>Thermo Scientific</td><td>09528101</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>A407P4</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss Axio Vision</td><td></td><td>Any fluorescent microscope with green filter will suffice</td></tr><tr><td>Hemacytometer</td><td>Fisher Scientific</td><td>0267152</td><td></td></tr><tr><td>Low melting point agarose</td><td>Promega</td><td>V2111</td><td></td></tr><tr><td>Microwave</td><td>Sears</td><td></td><td></td></tr><tr><td>Phosphate buffered saline, calcium free, magnesium free</td><td>HyClone</td><td>SH3025601</td><td></td></tr><tr><td>Power supply</td><td>BioRad</td><td>1645050</td><td></td></tr><tr><td>Refrigerator</td><td>Sears</td><td></td><td></td></tr><tr><td>Ruler</td><td>Staples</td><td></td><td></td></tr><tr><td>Slide</td><td>Fisher Scientific</td><td>12550143</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S5881</td><td></td></tr><tr><td>Sodium lauryl sarcosinate</td><td>Fisher Scientific</td><td>S529</td><td></td></tr><tr><td>Sybr green</td><td>Invitrogen</td><td>S7585</td><td></td></tr><tr><td>Tray</td><td>Fisher Scientific</td><td>15242B</td><td></td></tr><tr><td>Tris Base</td><td>Sigma</td><td>T6066</td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Trypsin</td><td>HyClone</td><td>SH3023601</td><td></td></tr><tr><td>Vortex</td><td>Fisher Scientific</td><td>02216108</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>154622Q</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay at JoVE.com

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

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 ...

assaying-dna-damage-in-hippocampal-neurons-using-the-comet-assay

Research

JoVE Journal

Neuroscience

27.3K Views.  University of Alabama-Birmingham. De komeet assay is een efficiënte manier voor het opsporen enkel-en dubbel-breuken, met inbegrip van alkali-labiele sites en DNA-DNA/DNA-protein cross-links op het DNA in alle cellen met inbegrip van hippocampale neuronen. De methode maakt gebruik van de differentiële migratie van DNA in een elektrisch veld als gevolg van verschillen in hoeveelheid DNA schade.

Video: Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

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

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

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 ...

Bioengineering

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

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

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Biology

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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 ...

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.

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 ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

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