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 border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<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></td>
			<td></td>
			<td></td>
			<td>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

29.2K 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

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 ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

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 ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

体外试验来测量磷脂酰乙醇胺甲基转移酶活

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...

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 ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

将细胞质Foci分类为哺乳动物应激颗粒的方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...