Name: comet assay as an indirect measure of systemic oxidative stress
Uploaded: 2015-05-22T15:00:00.000+00:00
Duration: 8 min 23 s
Description: Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

我们想感谢LHW基金会，特里森列支敦士登，为支持经济我们的研究氧化应激。

在巴塞尔大学进行的研究彗星试验，眼科批准了巴塞尔当地的伦理委员会 1.准备试剂<ol><li>制备的Dulbecco磷酸盐缓冲盐水（PBS）中（中Ca2 +，Mg ++的）加入PBS（1L包）和990毫升卫生署2 O至媒体溶液。将pH调节至7.4。存储在室温。 </li><li>制备溶胞溶液:制备千毫升在卫生署2 O.加入2.5 M氯化钠（146.1克），100毫摩尔EDTA（37.2克）和10mM Trizma碱（1.2克） </li><li>准备电泳缓冲液（300毫米氢氧化钠/ 1毫摩尔EDTA）加入30毫升10 M氢氧化钠和7.5毫升0.2M EDTA，QS到1500毫升卫生署2 O和拌匀。的总体积取决于该凝胶盒的容量。在使用之前，测量所述缓冲液的pH，以确保pH为&gt; 13。 </li><li>制备含0.4米的Tris缓冲液中和（48.5克加入到〜800毫升卫生署2 O，调节pH值至7.5），浓（＆＃62; 10 M盐酸）1000毫升卫生署2 O.存储溶液在RT。 </li><li>准备染色溶液。以溴化乙锭（EtBr的10×股票，20微克/毫升）加入10毫克的EtBr的在50毫升的dh 2 O和存储在RT。对于1X股票-将1毫升9毫升卫生署2 O.谨慎！处理溴化乙锭有足够的预防措施，因为它是一种已知的致癌物质。 </li></ol> 2.分离白细胞<ol><li>使用静脉穿刺获得的人类血液样品（20ml）中与抗凝血剂，如肝素。 </li><li>分开使用密度梯度细胞分离的淋巴细胞如的Histopaque。 <ol><li>简言之，将稀释血液在1:1的比例用PBS或RPMI（无FBS）和层600微升细胞分离器的液体。 </li><li>离心机1800 XG为20分钟，在4℃。吸出"血沉棕黄&#39;外衣成3-5毫升的PBS / RPMI中，并在300×g离心离心10分钟以沉淀淋巴细胞。 </li><li>从正上方PBS之间的边界检索淋巴细胞（RPMI）和细胞分离器的液体，使用移液器。从血浆和各管的细胞分离液层之间的界面除去白细胞频带，并收集到一个50ml管中。 </li></ol></li><li>使总体积至50ml用冷Dulbecco氏改良的Eagle培养基（DMEM）。在300×g离心洗细胞悬液三次用DMEM 10分钟。 </li><li>计数的总数目使用血球细胞。悬浮细胞在PBS中，小份到微量离心管中以10 7个细胞/管。 </li><li>吸管0.4毫升细胞到一个5毫升的塑料一次性管。加1.2毫升1％低胶凝温度琼脂糖在40℃。混合，并迅速与吸管1.2毫升细胞悬液到预涂覆载片的琼脂糖覆盖表面。避免产生气泡。 </li><li>允许琼脂糖凝胶进行约2分钟。是与用于胶凝的时间和温度保持一致，并确保琼脂糖浸没在裂解溶液之前完全建立。经过充分s等，淹没成裂解液在4〜ºC。 </li></ol> 3.裂解和电泳注意:所述的程序是用于pH&gt; 13的碱性条件下电泳。 <ol><li>后至少2小时，在〜4ºC，轻轻地从裂解溶液除去幻灯片。广场上滑动靠近一端的水平凝胶箱并排，滑动他们为接近越好。 </li><li>填充缓冲区水库和新鲜的pH值&gt; 13电泳缓冲液，直到液面完全覆盖的幻灯片（避免气泡在琼脂糖）。 </li><li>让载玻片坐在碱性缓冲液中20分钟，以允许该DNA的退绕和碱不稳定的损伤中的表达。 </li><li>打开电源到25伏（〜0.75伏/厘米），并调整到300毫安的电流通过提高或降低的缓冲水平。 Electrophorese幻灯片30分钟。 </li><li>关闭电源。轻轻抬起幻灯片从缓冲区和p蕾丝漏托盘。滴加大衣和缓冲液的幻灯片。允许的幻灯片坐至少5分钟。漏的幻灯片，并重复两次以上。 </li><li>染色用80微升1×溴化乙锭（EtBr的）的幻灯片和离开5分钟，然后浸在冷冻蒸馏水以除去过量染色剂。然后将盖玻片在幻灯片，并立即保存。商店幻灯片一个月在室温在干燥条件下。 注意:其他DNA污渍（ 例如 ，SYBR绿）也可以使用。注意！戴防护手套，因为大多数染料是致突变。 </li></ol> 4.定量DNA断裂<ol><li>以可视化的DNA损伤，观察一个40X的目标荧光显微镜下溴化乙锭染色DNA的幻灯片。 </li><li>通过测定DNA的迁移的长度和迁移的DNA（ 图1A）的百分比在细胞中定量评估的DNA损伤。定量DNA损伤（ 图1B）由Tail-时刻，橄榄MOMENT。尾矩，Olive -Moment定义先前已给予8-10。 注:根据我们的经验是不够的参数之一坚持。这些细胞通过设置自动化的工具，通过扫描显微镜载玻片和照片拍摄的细胞。 DNA断裂的定量可以通过图像分析软件包来完成。但是也有一些定制捕捉细胞的图像和量化的DNA损伤的几个不同的软件包。我们建议使用整个实验相同的软件包。化验员标识要打进电脑上的完整细胞或彗星，并用鼠标点击就可以了。彗星或完整的细胞，然后自动计分。 </li></ol>

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瑞典科学家Ostling和约翰松是第一个在自己的领域使用彗星试验来量化1细胞的DNA损伤。在中性条件下，这两个科学家使用，但是，只允许双链DNA断裂的检测。 Singh 等人后来适于测定使用在碱性条件下，这产生可能评估双和单链DNA断裂和检测碱不稳定的站点12中的敏感的版本。自其最初发展中，分析已修改在各种步骤以使其适用于评估类型在不同细胞类型13-15的DNA损伤。 与所有的技术，严格的支付重视技术细节是很重要的，以获得准确的结果16。根据我们的经验，得到最好的结果，如果每一个方法步骤，从溶液的制备彗星量化，通过专家的实验室进行TECHNicians。使用新鲜和正确准备的媒体，充足的移液技术，精确定时和（如前面的结果部分提到的）使用新鲜制备的白细胞为了裂解和凝胶电泳，以获得准确的结果至关重要的17。 在该试验中所有的各个步骤是为获得可靠的结果也同样重要。16在一般情况得到最好的结果，如果从溶液制备直到彗星量化每一个方法步骤是通过专家实验室technicians.Important执行似乎使用新鲜和正确地制备的培养基，充分移液技术，精确定时和如已经提到的结果部分的使用新鲜制备的白细胞为裂解和凝胶电泳以从测定17获得充分的效果。 由于做其他的方法，彗星试验技术都有其ADVAN每日新闻和缺点。作为一个敏感的方法，该法是脆弱的因素（ 如紫外线），这可能增加DNA断裂，从而影响效果。时，应避免可增强氧化应激，不同之处在于它是正在研究的任何因素。压力可以增加氧化应激不仅白细胞，而且在所有其它血液媒介和淋巴细胞的水平。如前所述有许多不同的和更直接的测量氧化应激18,19的方法。彗星试验是许多方法具有一定的优势之一。这些包括其相对的低成本，小数目必需的（&lt;10,000个细胞）的细胞和血液从而小样本每个病人所需的，时间的相对短的时间来量化细胞（约3天）的DNA损伤需要，其灵敏度和它的广泛的适用性，以在不同的细胞类型的驴的DNA损伤。在过去，我们选择与白细胞的工作，因为我们有经验，这细胞类型，它是适用于计划的具体研究。彗星测定法的另一个优点是，它可以用来检测不同类型和DNA损伤的水平，从而可以在研究其它各种领域之外的氧化应激，如DNA修复的研究中，补充试验或基因毒性研究应用。 氧化应激是目前公认的演奏在多种疾病的发病机理中起关键作用。彗星试验方法，同时测量氧化应激18,19的许多方法之一，是比较简单的，多功能的，价格低廉。当掌握，这种方法可以在医学的各个领域中，氧化应激作用8-10,20,21使用。

彗星试验最早是由瑞典科学家Ostling和约翰森1发达。的DNA是带负电荷的分子。电泳过程中，小，受损的DNA片段跑得更快走向带正电的阳极2。该DNA片段较小，向阳极更快的迁移，形成典型的"彗星"与"头"的完好，没有损坏DNA和一个"尾巴"受损的DNA片段3组成组成。受损DNA可以通过在主体4的不同机制，其保持DNA断裂相当平衡5的产生被修复。然而，如果平衡有利于DNA断裂，所述断裂会积聚并最终将有助于疾病的发展。 我们的DNA遭受约0.000165％的伤害，在给定的时间6。单链DNA断裂是指一个缺陷在DNA双螺旋的两条链中的一个而当DNA双螺旋的两条链被破坏双链DNA断裂引起的。有多种因素可促进DNA断裂在给定的时间量，如年龄细胞，香烟烟雾，各种药物或氧化胁迫7-9。如果导致增强的DNA断裂的所有因素保持不变，那么DNA断裂彗星试验的装置的定量是氧化应激的良好间接参数。 今天使用越来越多的医学眼科，包括彗星实验的方法。其中一个原因是，氧化应激是越来越多的认可作为发病机制各种疾病8-11和彗星试验是一种方法与氧化应激可以间接进行量化。

<table><tbody><tr><td>Dimethylsulfoxide (DMSO)</td><td>Qualigens</td><td>(CPW59)</td><td></td></tr><tr><td>Disodium EDTA</td><td>HiMedia</td><td>(RM1370)</td><td></td></tr><tr><td>Ethidium Bromide</td><td>Sigma</td><td>(E-8751)</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>(1077-1)</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) (Ca++, Mg++ free)</td><td>HiMedia</td><td>(TS1006)</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Ranbaxy Rankem</td><td>(S0160)</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>BDH-Merck</td><td>(89021)</td><td></td></tr><tr><td>Triton X-100</td><td>HiMedia</td><td>(RM 845)</td><td></td></tr><tr><td>Trizma Base</td><td>Spectrochem</td><td></td><td></td></tr><tr><td colspan="4">Materials required for gel electrophoresis</td></tr><tr><td>Normal Melting Agarose (NMA)</td><td>HiMedia</td><td>(RM273)</td><td></td></tr><tr><td>Low Melting Point Agarose (LMPA)</td><td>Sigma</td><td>(A9414)</td><td></td></tr><tr><td>Methanol - Qualigens</td><td></td><td></td><td></td></tr><tr><td>Coverslips (No. 1, 24 x 60 mm)</td><td>Blue Star</td><td></td><td></td></tr><tr><td>Microcentrifuge Tubes</td><td>Tarsons</td><td>(500010)</td><td></td></tr><tr><td>Micropipettor and Tips</td><td>Tarsons</td><td></td><td></td></tr><tr><td>Microscope Slides, Conventional / Micro gel electrophoresis (MGE) slides</td><td></td><td></td><td></td></tr></tbody></table>

comet assay as an indirect measure of systemic oxidative stress

高等真核生物的生活不能没有氧气;然而，矛盾的是，氧气可以对他们有害的。氧分子在化学上是相对惰性的，因为它具有位于不同圆周率*反键轨道2未成对电子。这两个电子具有平行自旋，这意味着它们在围绕自身的轴相同的方向旋转。这就是为什么氧分子不是非常活泼。氧的活化可以由两个不同的机制发生;无论是通过经由一个电子还原的时间（一价降低），或通过足够的能量吸收扭转不成对电子的一个的旋转。这导致产生活性氧物质（ROS）的。有多种方法，其中人体消除活性氧在其生理状态。如果ROS产生超过修复能力，氧化应激的结果和损害不同的分子。有许多不同的方法，通过该氧化应激可以是我asured。这个手稿的重点是名为细胞凝胶电泳，也被称为"彗星试验"，它允许DNA断裂测定的方法之一。如果已知会导致DNA损伤，比氧化应激其它所有因素保持不变，DNA损伤的彗星试验测定的量是氧化应激的良好参数。原理是简单的，并依赖于一个事实，即DNA分子是带负电荷。一个完整的DNA分子具有其不电泳期间迁移这样一个大的尺寸。 DNA断裂，但是，如果存在导致较小的片段中的电场移向阳极。更小的片段迁移更快。作为片段具有不同尺寸的电泳的最终结果是不是一个独特的线，而是以一个彗星的形状的连续体。该系统允许在小区中的DNA断裂的产生的"彗星"，从而对一个量化。

虽然彗星测定法的方法呈现可再现的结果，它可以由多种因素的影响。一个这样的因素是是否白细胞之前裂解和凝胶电泳或是否该步骤执行对新鲜制备的白细胞冷冻保存。 图2显示，在第2组，其中白细胞前体裂解和凝胶电泳SS- DNA冷冻保存符是显著高，比第1组，其中裂解和凝胶-electrophoresis已于新鲜制备细胞完成的。 该分析还可以用来间接评估系统性氧化应激。表1示出了描述性统计Tail-时刻和Olive-时刻在吸烟者和健康受试者。这些数据表明，吸烟半包香烟每天超过一倍的单链DNA断裂的循环白细胞9。 <img alt="图1A" src="/文件/ ftp_upload / 52763 / 52763fig1A.jpg"/&gt; <img alt="图1B" src="/files/ftp_upload/52763/52763fig1B.jpg" /> 图1（a）显示在彗星试验分析中使用的显微镜。 （B）Δ描述了一个典型的"彗星"有一个光明的头和尾（小受损的DNA片段）。 <a href="https://www.jove.com/files/ftp_upload/52763/52763fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52763/52763fig2.jpg" /> 图2.单链DNA断裂是显著更高时白细胞被冷冻保存（第2组）相比时新鲜制备白细胞前体裂解和凝胶电泳使用。 <imgALT ="表1"SRC ="/文件/ ftp_upload / 52763 / 52763table1.jpg"/&gt; 表1.吸烟者相比，年龄和性别的单链DNA的两倍以上数量突破相匹配的健康非吸烟者。

Watch this Scientific Journal Video about Comet Assay as an Indirect Measure of Systemic Oxidative Stress at JoVE.com

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

彗星试验测定DNA断裂，诱发不同的因素。如果所有的因素（除氧化应激）引起DNA损伤被保持恒定，DNA损伤的量是氧化应激的良好间接参数。这个协议的目标是使用彗星测定氧化应激的间接测量。

彗星试验系统性氧化应激的间接测量

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

comet-assay-as-an-indirect-measure-of-systemic-oxidative-stress

Research

JoVE Journal

Biology

29.3K 次观看.  University of Basel. 彗星试验测定DNA断裂，诱发不同的因素。如果所有的因素（除氧化应激）引起DNA损伤被保持恒定，DNA损伤的量是氧化应激的良好间接参数。这个协议的目标是使用彗星测定氧化应激的间接测量。 

视频: Comet Assay as an Indirect Measure of Systemic Oxidative Stress

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.

利用彗星试验评价体外DNA 损伤

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 ...

科研

癌症研究

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.

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

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

生物工程

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

斑马鱼的响应行为的检测，以测量光强的变化

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

生物学

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.

在彗星分析中使用冷冻组织评估DNA损伤

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 ...

遗传学

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.

彗星试验测定DNA损伤的海马神经元

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 ...

神经科学

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

应用的<em在体外</em&gt;保护DNA检测，以可视化应力调解属性的DPS蛋白质

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

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.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

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.

一种用于评估细胞DNA损伤的高通量彗星检测方法

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

To begin, prepare 0.6% low melting point agarose in PBS, and place it in a water bath at 37 degrees Celsius. Labeled the frosted end of the pre-coated slides with name, date, and treatment information using a permanent marker or pencil.
Place a chilling plate on a flat bench, and insert two frozen cooling packs into the sliding drawer below the metal surface. Then, place the slides on the chilling plate to pre-chill for 1 to 2 minutes. Centrifuge and remove the supernatant from the sample tubes, and place them immediately back on ice. Resuspend the cell pellet with 200 microliters of 0.6% low melting point agarose, and mix by pipetting.
Next, add 80 microliters of agarose-containing cells onto a chilled slide, and quickly place a coverslip on the gel. Allow the gel to set on the chilling plate for 1 to 2 minutes. In the meantime, prepare 500 milliliters of working solution of lysis buffer, and pour it into the lysis dish.
After the gels have set, remove the coverslips quickly by gently holding the slide between thumb and forefinger, and sliding the coverslip off the gel. Then, place the slides inside a slide carrier, with all the black dot marks on the slides facing in the same direction. Place the slide carrier inside the lysis dish. Close the lid of the lysis dish, and put it for incubation.
Carefully remove the slide carrier from the lysis dish without disturbing the gels. Gently, place the slide carrier in a washing dish pre-loaded with ice-cold double-distilled water. Ensure that the slides are completely submerged, and leave the setup for 30 minutes.
Place a frozen cooling pack under the electrophoresis tank to maintain optimal buffer temperature. Then, add ice-cold electrophoresis working solution to the tank, and transfer the slide carrier into it. Orient the slides, such that the cell containing gels point toward the cathode.
Incubate the slides in the electrophoresis tank for 20 minutes, to allow the DNA to unwind. Then, turn on the power supply to perform electrophoresis for 20 minutes at 1.19 volts per centimeter. After the electrophoresis is complete, turn off the power supply, and remove the slide carrier from the electrophoresis tank. Allow the slide carrier to drain on tissue paper for 30 seconds.
Next, place the slide carrier into a dish containing neutralization buffer, and leave it for 20 minutes. Following this, place it in a washing dish containing ice-cold double-distilled water, and leave it for 20 minutes. After washing, remove the slide carrier from the water, and allow the slides to dry in an incubator at 37 degrees Celsius for 1 hour.
To rehydrate the slides, transfer the slide carrier to a washing dish containing ice-cold double-distilled water and leave it for 30 minutes. Then, place the slide carrier into a staining dish containing 2.5 micrograms per milliliter propidium iodide solution.
Close the lid of the staining dish, and incubate it for 20 minutes in the dark at room temperature. After incubation, transfer the slide carrier to a separate dish, and wash it with ice-cold double-distilled water for 20 minutes.
Remove the slide carrier from the dish, and dry it completely in the dark, either in a 37 degrees Celsius incubator or at room temperature. After the slides dry completely, store them in a slide box in the dark until ready for image analysis.

用于细胞中 DNA 损伤检测的 Comet 检测

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Thiobarbituric Acid-Reactive Substances Assay to Assess Oxidative Stress in Human Serum

Label the glass tubes for the samples to be analyzed, then, add 100 microliters of the prepared sample to each tube. Add 200 microliters of 8.1% SDS to each sample, and gently swirl the glass tube in a circular motion to mix.
Add 1.5 milliliters of 3.5 molar sodium acetate buffer to each sample. Then, add 1.5 milliliters of aqueous 0.8% thiobarbituric acid solution. Bring the final volume in each tube to 4 milliliters by adding 700 microliters of deionized water. Tightly cap the glass tubes, and incubate them in a heating block set to 95 degrees Celsius for 1 hour. Cover the tubes with aluminum foil to prevent condensation. Then, remove the tubes from the block and incubate them on ice for 30 minutes.
After the incubation, centrifuge the samples and standards at 1,500 x g for 10 minutes at 4 degrees Celsius. Transfer 150 microliters of supernatant from each tube to a well in a 96-well plate, removing any air bubbles with a pipette tip.
Measure the absorbance of the samples at 532 nanometers. Then, subtract the average absorbance reading of the blank samples from all other absorbance readings.
Create a standard curve and use it to calculate unknown sample concentrations.