该研究是由加拿大卫生部基因组学研究和发展倡议（GRDI）和加拿大监管策略生物技术（华润雪花）。作者有没有利益冲突。

确保所有牧，住房，处理和实验遵守当地的法律和法规，而且实验的协议之前，开始任何研究批准。对于所描述的实验中，动物护理，处理和治疗批准了加拿大卫生署动物管理委员会。见“试剂表”，供应商的信息。 1.采集生物样品<ol><li>细胞或动物组织<ol><li>生长在含有10％胎牛血清，青霉素100单位/ ml和链霉素100μg/ ml的F12-K媒体人肺泡腺癌A549细胞。 </li><li>种子细胞在每一个10厘米的板约100万细胞。对于每个试验，使用一组12片在总（每一种生物复制x四剂三个板），以提供足够的DNA（80微克），用于酶促消化并HPLC分析。 </li><li>当细胞密度达到大于约imately板面面积的70％，除去培养基并用4ml磷酸盐缓冲盐水（PBS），pH 7.4中洗涤细胞两次。 </li><li>用于培养的动物细胞，可以使用溴酸钾3作为阳性对照。 注：溴酸钾3浓度和8-氧dGuo频率之间的正线性关系已在文献中报道9。 </li><li>在PBS溶解溴酸钾3。确保了在细胞培养基中的终浓度大于1毫米，以获得在-8-氧代dGuo相对于未暴露的细胞中有统计学显著增加。添加溴酸钾3同样浓度系列中的（ 例如 ，12 10厘米板）每块板。 </li><li>孵育板3小时，在37℃。取出纸张，用PBS洗一次。 </li><li>加入1毫升的胰蛋白酶溶液（原液浓度为2.5g / ml）和孵育3分钟，在37℃。 </li><li>用4毫升的PBS洗涤，收集每个设置成一个50ml锥形polystyr细胞烯离心管中，并离心在1000×g离心5分钟，在4℃。 </li><li>除去PBS并保存细胞沉淀在-80℃直至进一步分析。人造DNA的氧化可以通过核孤立的进一步减少，因为其他地方的10说明。 </li></ol></li><li>动物组织<ol><li>由于所需剂量的动物。对于这个协议，成年（10周龄），每日治疗男性木塔鼠标口服灌胃，连续三天20毫克DBC /千克体重每天溶于橄榄油。 注：该转基因动物港口工程λ噬菌体和突变LacZ报告基因从大肠杆菌大肠杆菌 11用于转基因啮齿动物突变分析12。 </li><li>执行动物尸检例如，麻醉小鼠可以使用异氟醚，然后通过颈椎脱位其次是胸腔开放安乐死。立刻闪冻组织在液氮中。商店组织在-80℃直至分析。 注：安乐死的处理后的定时可以是另外一个重要的变量（例如，DNA的氧化是最大的在大鼠脑和肝13噪音暴露后72小时）。 </li></ol></li></ol> 2. DNA提取，沉淀和冲洗（对于组织直接进行2.2） <ol><li>均化收集的细胞在50ml锥形聚苯乙烯离心管中，用1ml的DNA分离剂如DNAzol。使用一个1000微升枪头驱散细胞沉淀，直到该溶液均匀。转移匀浆到一个新的1.5 ml离心管。在冰上孵育10-20分钟。继续执行2.3。 </li><li>均质15-20 mg组织中含有500微升的DNA隔离剂1毫升手持不粘玻璃匀浆。轻轻抬起，并约1分钟下均质;较软的组织会需要更少的时间。温和的治疗，确保DNA的较小的剪切。储存在冰匀浆约10-20分钟。 </li><li>丸匀浆离心10,000×g离心10分钟，并在1.5毫升锥形离心管中4℃。 </li><li>将得到的上清液小心转移到一个新的1.5 ml离心管仔细注意避免接触沉淀。 </li><li>通过加入0.5ml的100％EtOH中每1ml匀浆沉淀从匀浆的DNA。倒置试管10次，以确保分离试剂和EtOH充分混合。 </li><li>在这一点上，DNA的沉淀物为粘稠，线轴它到塑料枪头（ 如 ，用200μl的最大保持容量），然后转移到一个新的1.5毫升锥形离心管中。 注意：快速离心可以用来从分离的DNA除去任何剩余的溶胞产物。 （DNA洗涤和增溶） </li><li>加入1毫升75％乙醇的分离的DNA。反相管10倍彻底暂停DNA沉淀。 </li><li>小心倒出从管的乙醇。确保日ê的DNA沉淀粘在管的一侧。存储管垂直1-2分钟，并使用移液管从管底除去任何过量的乙醇。 </li><li>重复该DNA洗一次以上，并且无论在EtOH样品储存在-20℃（稳定数个月），或立即进行消化。 </li><li>溶解DNA的消化缓冲液（如下所述），然后量化使用标准光谱方法（ 即 ，260nm处的吸光度使用的NanoDrop分光光度计）。 </li></ol> 3.酶消化<ol><li>制备“消化缓冲液”通过组合适当体积（取决于样品的数量被消化）的50mM的磷酸二氢磷酸钠（含有2.0毫米氯化钾，1.0mM的desferal（DFO）和200毫米氯化镁2）用50mM二元磷酸钠（还含有2.0毫米氯化钾，1.0mM的DFO和200mM的MgCl 2的）。每个样品需要4.0微升一元和17.0微升磷酸氢溶液。 </li> &lt;李&gt;从管和风干样​​品的底部去除任何残留的EtOH为约5分钟。确保DNA不完全风干。 </li><li>解散80微克提取的DNA 21.0微升消化缓冲液中，加入1单位的DNase I溶解在2.0微升消化缓冲。 </li><li>涡轻轻实现充分混合和孵育1.5小时，37℃。 </li><li>在温育期结束时，添加216.4微升二元50含有2.0毫米氯化钾，1.0mM的DFO和200mM的MgCl 2的毫磷酸钠溶液。 </li><li>制备“消化缓冲-2”通过组合的消化缓冲液一体积与九卷二元的，50毫磷酸钠（2.0毫米氯化钾，1.0mM的DFO和200mM的MgCl 2的）。 </li><li>添加0.025单位的磷酸二酯酶（PDE）我酶16.3微升消化缓冲-2。移液器上下几秒钟，然后涡旋轻轻实现彻底混合并孵育1.5小时，在37℃。 </li><li>添加00.4单位碱性磷酸酶（AP）在8.0微升的消化缓冲-2。移液器上下几秒钟，然后涡旋轻轻实现彻底混合并孵育1.5小时，在37℃。添加33.0微升HPLC级甲醇。 </li><li>运行消化的DNA样品在HPLC上，如下面所述，马上或储存在-80℃直至使用，以减少氧化。注：这里建议是基于这样的间隔1.5小时的消化步骤其他9和研究结果表明，类似的协议实现完整的DNA消化9。因此，它被认为是唯一的限制因素，以实现完全的消化时间和酶抑制和样品的不均匀性可以忽略不计。 </li></ol> 4. HPLC运行：流动相，仪器仪表安装和维护的制备<ol><li>用超纯水制备所有的缓冲液和具有高粘结强度树脂（ 如 CHELEX 100治疗：苯乙烯divinylbe亚氨基二乙酸与共聚物nzene离子与高亲和力螯合过渡金属），以尽量减少样品制备过程中金属污染物和DNA氧化。 </li><li>制备250mM的磷酸钠缓冲液，pH 6.2。使用的比例为1：3.6957的磷酸氢二钠（FW =177.99克/摩尔），以磷酸二氢钠（FW =119.98克/摩尔）。 实施例：因此，对于一个3升原液结合70.81克的一元和28.43克二元粉末，加水到例如，2.8升，并验证该溶液的pH为6.2。调节pH值用250毫单或磷酸氢二钠溶液，分别地和不准备浓缩的酸或碱。添加2.24克氯化钾，得到10mM的氯化钾的浓度。 </li><li>制备流动相在三瓶，至少1 L各自含有：溶剂B - HPLC级甲醇，溶剂 - B超纯水和溶剂C - 从步骤4.2的磷酸盐缓冲液。将高效液相色谱流动相供电管线入瓶; remaini毫微管也必须浸在瓶（ 例如，溶液C）之一，即使它们不需要混合底漆。 </li><li>在运行过程中，混合的流动相溶液作为6％溶剂A，74％溶剂B至20％溶剂C实现的50mM磷酸钠缓冲液（pH 6.2）与2.0毫米氯化钾，和6％的MeOH最终溶液。 </li><li>从电化学检测器分离的电极，拆解和干净的参考电极和工作电极与电极清洗溶液和制造商建议的抛光盘。冲洗清洁的表面用超纯水，轻轻将水擦干，并保证检测是转向系统之前干。 </li><li>重新组装检测器，流动相入口连接到电极。打开的流动和允许流动相填满电极附接出口流动相管之前。安装前的优化开始一个新列。请确保安装在正确的方向和follows所有制造商的建议。 </li><li>打开仪器HLPC样品遥遥领先（ 例如，1小时）进行分析，以便平衡并减少基线噪音。请参阅表1中建议的设置;背压极限应设置为3000 psi至避免色谱柱损坏。打开真空脱气机。继续吸泵和设置溶剂梯度。 </li><li>干素根据制造商的说明泵。为此，当一个流动相已被替换或油管的入口暴露于空气中以润湿线。 </li><li>找到启动光盘，然后将10-15毫升的塑料注射器在那里。确保注射器打开前行完全插入。要打开行简单地把盘逆时针的一个转机。 </li><li>开始用适当的HPLC接口选择的素数。轻轻拉动注射器将流体抽入的行。一旦流体流动，首要是完整的;允许程序完成运行。重复所有行（通常是四个，取决于仪器）。 </li><li>或者，当线已经从早期的干素润湿湿素（见下文）。 注意：当大量的时间用途（ 例如 ，2周或更多）之间已经过去时，以干质被推荐。 </li><li>流动相溶剂成分的组合物改变为每个使用的HPLC用户界面的25％。从界面中，选择“直接功能”选项，然后选择“湿总理”选项。这将素都在同一时间的行。 </li><li>确保所有空气从系统中通过观察湿黄金线在进入废物容器。好后素，确保没有气泡被留在该行。如果泡沫依然存在，重复吸。 </li><li>一旦泵启动，开始移动的HPLC流动相。使用的HPLC接口向移动相的组成手动更改至6％甲醇（溶剂A）和94％的水（溶剂B），并选择为0.9毫升/分钟的流速。允许5分钟以清除任何来自甲醇塔清洗步骤。 </li><li> 5分钟后，改变组合物中以6％甲醇（溶剂A），74％水（溶剂B），20％缓冲液（溶剂C），增加的流速为1.0毫升/分。如归纳于表1，并设置背压限制到3000 psi至避免柱损坏设置其他参数。 注：潜在的问题与HPLC设置，包括基线漂移，分裂峰减弱信号强度。这些通常可以通过清洗列中的每个使用，频繁电极清洗后，并确保缓冲溶液是无污染来克服（ 例如 ，颗粒物质或微生物生长）。 </li><li>运行流动相为约1-1.5小时，从而实现稳定的基线信号。 </li><li>使用仪器的软件接口，如表1所示，并设定运行时间为15分钟，选择参数。注入SAMPLE。使用增加的运行时间复杂样品（通过观察峰值的存在或不存在超出15分钟间隔测定该凭经验）。 注：仪器软件允许样品运行情况的编程和设置自动进样。 </li><li>的运行已完成之后，使用该检测器的接口关闭的检测器单元。遵循制造商的建议有关的检测器处理不当断电和关闭会损坏仪器是很重要的。不要通过切换器的背面，因为这可能会损坏仪器无法关闭电池。 </li><li>手动改变流动相组合物至6％甲醇和94％的水。运行5分钟。 </li><li>手动降低流速0.7毫升/分钟，紧接在组合物改变至50％MeOH和50％的水。运行至少20分钟。失败运行中推荐的时间，可能会损坏到柱和检测器这一步。关闭流，一个ð然后关闭真空脱气机。 </li></ol> 5.编写标准<ol><li>制备流动相缓冲液为在上述步骤4.1-4.3所述。使用的HPLC流动相配制标准，以避免来自不同的解决方案的样品注入后混合所得的峰是很重要的。 </li><li>稀释此溶液，五分之一的超纯水在使用前，而最终的溶液必须含有6％甲醇。 </li><li> dGuo标准 注意：需要的dGuo检测高氧化电势可以增加测量干扰电化合物的风险。这可以通过验证，例如，UV检测dGuo的在260nm处的标准曲线，并比较它与EC检测所创建的标准曲线。如果两个对准孔（ 例如 ， 补充图1）和样品基体效应已被考虑进去，人们可以在260处继续进行dGuo检测通过UV，而不是EC检测。 <ol><li&gt;衡量一个1.5 ml离心管1.0毫克dGuo的。添加1.0毫升HPLC级甲醇。 </li><li>涡高速，直到dGuo完全溶解。这是主要的股票;储存在-20℃下进行数周。 </li><li>创建辅助dGuo股票（0.5毫米），稀143微升的主要dGuo股票在857微升HPLC流动相。储存在冰，直到使用，并准备每天新鲜。 </li><li>作进一步稀释来创建标准曲线（ 例如 ， 表2）。冰上存储解决方案，并准备每天新鲜。串联实验样品运行标准。 </li><li>在此之前运行，使用不同股票的最高浓度，以找到最佳的电压检测器的电压。 </li></ol></li><li> -8-氧代dGuo标准<ol><li>测量1.0毫克8-氧dGuo在1.5ml微量离心管中。添加0.1毫升HPLC级甲醇。涡流在高速直到-8-氧代dGuo完全溶解。这是-8-氧代dGuo主存量。储存在-20℃下SEVERAL周。 </li><li>在一个单独的试管中，结合2.9微升的主要股票和997.1微升的高效液相色谱流动相缓冲液，旋涡。这是次要的股票（10,000纳米）。 </li><li>要创建标准曲线（250 nm）的有效的解决方案，结合24.4微升二次股和975.6微升流动相缓冲。 </li><li>执行进一步的稀释，以提供创建标准曲线所要求的解决方案（ 例如 ， 表3）。冰上存储解决方案，并准备每天新鲜。与实验样品运行标准。 </li></ol></li><li>定量<ol><li>曲线下整合的区域为两个-8-氧代dGuo和dGuo使用标准软件（作为液相色谱-计算机接口的一部分;见补充图2A）。 </li><li>构建标准曲线（每种分析物与面积的曲线（ 补充图2B）下的已知浓度。使用标准曲线的方程，计算出的比率为8-氧dGuo / dGuo每个样品。 </li></ol></li></ol> 6.琼脂糖凝胶电泳注：琼脂糖凝胶电泳可被执行以验证DNA酶切的完整性。 <ol><li>制备50倍Tris-乙酸-EDTA（TAE）缓冲液，通过在750ml超纯水中溶解242克Tris碱（FW = 121.14）。添加57.1毫升冰醋酸和100ml的0.5M EDTA（pH值8.0; EDTA会完全溶解时将溶液pH调节至8.0与如氢氧化钠）和超纯水加至1升商店的终体积在RT，稀释使用前该溶液50倍。 </li><li>称量1g琼脂糖和溶解在100ml 1×TAE缓冲液，加热，在微波1-2分钟。戴上护目镜和防护设备以避免与沸腾的琼脂糖接触。 </li><li>冷却液，直到约50℃，加入5微升溴化乙锭（注意：诱变剂），倒入琼脂糖凝胶运行装置。插入梳子。</li><li>等到溶液凝固，倒TAE缓冲液在凝胶和装载样品（体积取决于梳大小;通常情况下，10-20微升DNA样品装载，与6X运行染料混合，可视化和方便装）。 </li><li>运行凝胶直至染料迁移的凝胶的约2/3的长度（ 例如 ，45分钟，在150V）。可视化下的紫外线DNA条带。 </li></ol>

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No conflict of interest or competing financial interests declared.

虽然-8-氧代dGuo已被报告为脱氧核糖核酸氧化的有用的生物标志物，其可靠的量化可以构成挑战。虽然有几种公开的方法存在，有必要对协议的全面的，描述概述，以允许研究人员部署在他们的实验室的方法。这里，我们提出的HPLC为基础的协议，将允许新用户建立一种有效的方法-8-氧代dGuo检测和定量的详细概述。 已经描述了8个氧代dGuo量化三种主要的方法。这些包括酶联免疫...

活性氧（ROS），其稳态水平期间许多病理学病症和xenotoxic代谢能增加，有助于氧化性DNA损伤的频率增加。在几个可能的核碱基的氧化产物，氧化性DNA损伤可容易地使用稳定标记测量-8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧dGuo），它是2的氧化形式之一&#39; -deoxyguanosine（dGuo）1。 -8-氧代dGuo是最丰富的DNA损伤2，因此，已研究以更详细的DNA氧化标志物尽管多种DNA氧化产物3的存在。在人类中，这种损伤可以通过碱基切除修复了8羟基鸟嘌呤糖苷酶1（hOGG1基因）4修复。如果左未修理，8-氧dGuo可以向形成的碱基对替代突变（ 即 ，G到T颠换）4。重要的是，8-氧dGuo是一个既定的标记FO- [R DNA损伤有关的启动和促进癌变2。因此，8-氧代- dGuo准确定量是氧化性DNA损伤5的有用和理想的生物标志物。 人们普遍混乱关于对氧化损伤的形式的2-脱氧鸟苷，此外，化合物（S）的正确名称常规测量作为氧化性DNA损伤6的生物标志物的正确名称的文献。的8-氧dGuo（ 图1中示出）的6,8-二酮和6-烯醇，8-酮互变异构形式都在文献5,7中讨论的两个最突出的互变异构体。的6,8-二酮的形式是在7.4的生理pH值的最突出的形式，并且是最突出的DNA氧化产物7。因此，8-氧dGuo，而不是8羟基dGuo是用于该氧化产物6的最适当的名称。同样重要的是要注意，2-脱氧鸟苷（dGuo），而不是nucleob酶鸟嘌呤（卦）或核糖鸟苷（国），分别是由大多数方法6检测。 精确检测和8-氧代dGuo量化是具有挑战性的，原因是：在该DNA样品的消化ⅰ）变异，ⅱ）dGuo的不定氧化成8-氧dGuo可能发生样品制备过程中，和iii）需要在分析HPLC-ED法8验证有效。在这个协议中，我们通过提供条件，有利的完整的DNA消化和ii）通过包含金属螯合剂和螯合剂处理的溶液和一种特殊的DNA分离试剂，而ⅲ）仅部分通过包含寻址旨在实现ⅰ）阳性对照，从而提供了该方法能够检测-8-氧代dGuo生物样品中。进一步验证超出了本文的范围。但是，我们相信，这一协议将有助于准用户确定在何种程度上他们需要正式验证协议，这取决于它们的目的。所需的方法的正式验证步骤的清单还设置。在8氧dGuo检测方法的开发和部署，公布的方法进行了回顾和巩固。因此，该方法消除了需要从几个公布来源往往缺乏重要的实验细节，同时还提供测试快速和简单的方式，如果该方法用于-8-氧代dGuo的检测和定量已成功地采用了收集信息。这种适应方法采用成功地分析DNA样本培养细胞和小鼠组织。这个视频文章将有助于其他群体建立一种有效的方法可靠的检测和8-氧dGuo定量用HPLC-ED。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>8-oxo-dGuo standard</td>
			<td>Cayman Chemical Company</td>
			<td>89320</td>
			<td>Inappropriately referred to as &#34;8-hydroxy-2&#39;-deoxy Guanosine&#34; - see Fig. 1 and text for details</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P5931</td>
			<td>From E.coli</td>
		</tr>
		<tr>
			<td>Chelex 100</td>
			<td>Sigma-Aldrich</td>
			<td>C7901</td>
			<td>Chelates heavy metals</td>
		</tr>
		<tr>
			<td>Desferoxamine mesylate</td>
			<td>Sigma-Aldrich</td>
			<td>D9533</td>
			<td></td>
		</tr>
		<tr>
			<td>dGuo standard</td>
			<td>Sigma-Aldrich</td>
			<td>D7145</td>
			<td></td>
		</tr>
		<tr>
			<td>Dibasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9390</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon sperm</td>
			<td>Sigma-Aldrich</td>
			<td>D1626</td>
			<td>Sodium salt</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4527</td>
			<td>TypeII, from bovine pancreas</td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Invitrogen</td>
			<td>10503-27</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E4884</td>
			<td>The compound would not completely dissolve until solution pH is adjusted to 8.0 with e.g. NaOH</td>
		</tr>
		<tr>
			<td>F12-K media</td>
			<td>ATCC</td>
			<td>30-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Guard column</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 0204GC</td>
			<td>Protects colum from contamination; may also lead to pressure build-up</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Monobasic sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Invitrogen</td>
			<td>15190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphodiesterase I enzyme&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3243</td>
			<td>Type II from Crotalus adamaneus venom</td>
		</tr>
		<tr>
			<td>Teflon homogenizer</td>
			<td>Thomas Scientific</td>
			<td>7724T-1 or 7724T-5 for 1 or 5 mL, respectively</td>
			<td>Volume (holding capacity) depends on the amount of sample to be processed.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Invitrogen</td>
			<td>15050-065</td>
			<td></td>
		</tr>
		<tr>
			<td>YMC-BASIC column with bonded spherical silica</td>
			<td>Chromatographic Specialties</td>
			<td>YBA 99S03 1546WT</td>
			<td></td>
		</tr>
	</tbody>
</table>

hplc measurement of the dna oxidation biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in cultured cells and animal tissues

氧化应激与许多生理和病理过程，以及生物外源性代谢相关，导致生物大分子的氧化，包括DNA。因此，高效的检测DNA氧化是多种学科的研究，包括药物和毒物的重要。氧化损伤的DNA的一个共同的生物标志物是-8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧代dGuo;经常误称为8-羟基-2&#39;-脱氧鸟苷（8-OH-dGuo或8氧代dG的））。对于-8-氧代dGuo测量用高压液相色谱电化学检测器（HPLC-ED）几个协议已被描述。然而，这些主要适用于具有促氧化剂处理纯化DNA。此外，由于实验室之间方法上的差异，主要是由于在分析设备的差异，采用用于检测-8-氧代dGuo通过HPLC-ED公布的方法需要由每个实验室仔细优化。一个全面的协议，描述这样一个优化过程，是缺乏。这里，一个详细的协议是从培养的细胞或动物组织中所述的8-氧dGuo通过HPLC-ED的检测，在DNA中。它说明了如何DNA样品制备能够容易且快速地优化，以尽量减少可能发生的样品制备过程中不希望的DNA的氧化。这个协议显示了如何检测-8-氧代dGuo与氧化剂溴酸钾3处理培养的人肺泡腺癌细胞（ 即 ，A549细胞），以及从暴露在多环芳烃二苯并（ 闪避，p）的屈小鼠的脾脏（DBC，前身为二苯（A，L）芘，DALP）。总体来说，这一工作说明了如何用HPLC-ED方法可以用于-8-氧代dGuo生物样品中的检测可以容易地优化。

dGuo观察到有4.7分钟而-8-氧代dGuo具有约6.4分钟（ 图2A和B）的保留时间，保留时间。有一个在两种分析物之间的峰高约1000倍的差异，如在图2C中看到的。伏安-8-氧代dGuo和dGuo通过运行标准在+0.2的范围内的工作电位，获得至1.1 V的最佳工作电位为-8-氧代dGuo被确定为+ 0.5V，和0.9 v对于dGuo（ 图3）。这些电位是与文献14,15中所述的其它玻璃碳电极?...

Watch this Scientific Journal Video about HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2â€™-deoxyguanosine, in Cultured Cells and Animal Tissues at JoVE.com

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&amp;#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

该协议的目的是将DNA氧化标志物的检测，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷（8-氧dGuo）通过HPLC-ED，从培养的细胞或动物组织的DNA。

该DNA氧化标志物的高效液相色谱法测量，8-氧代-7,8-二氢-2&#39;-脱氧鸟苷，在培养细胞和动物组织

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

hplc-measurement-dna-oxidation-biomarker-8-oxo-78-dihydro-2

Research

JoVE Journal

Chemistry

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

26.1K 次观看.  Health Canada. 该协议的目的是将DNA氧化标志物的检测，8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧dGuo）通过HPLC-ED，从培养的细胞或动物组织的DNA。 

视频: HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

稳态，稳定前的状态，单营业额动力学测量DNA糖基化酶活性

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

科研

化学

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-基于化验，以监测外核苷酸/核苷代谢的人类慢性淋巴细胞白血病细胞

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

确定缓蚀剂/金属界面的化学成分与XPS：最小化后浸入氧化

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

光子温度计的制作与测试

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

同时测量 HDAC1 和 HDAC6 活动在 HeLa 细胞使用 UHPLC 女士

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

动态和电泳光散射的 DNA-磁性颗粒结合分析

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

解析高强共聚物芳纶纤维使其力学性能的测定

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

[11C_SNAP-7941] 自动合成和实时动力学评估的技术方面

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Quantitative Atomic-Site Analysis of Functional Dopants/Point Defects in Crystalline Materials by Electron-Channeling-Enhanced Microanalysis

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident high-energy electron beam is rocked with the submicrometric pivot point fixed on a specimen. This method enables us to quantitatively derive the site occupancies and site-dependent chemical information of impurities or intentionally doped functional elements in a specimen, using energy-dispersive X-ray spectroscopy and electron energy-loss spectroscopy attached to a scanning transmission electron microscope, which is of significant interest to current materials science, particularly related to nanotechnologies. This scheme is applicable to any combination of elements even when the conventional Rietveld analysis by X-ray or neutron diffraction occasionally fails to provide the desired results because of limited sample sizes and close scattering factors of neighboring elements in the periodic table. In this methodological article, we demonstrate the basic experimental procedure and analysis method of the present beam-rocking microanalysis.

We provide a general outline of quantitative microanalysis methods for estimating the site occupancies of impurities and their chemical states by taking advantage of electron-channeling phenomena under incident electron beam-rocking conditions, which reliably extract information from minority species, light elements, oxygen vacancies, and other point/line/planar defects.

通过电子引导增强微分析对晶体材料中功能性多裤/点缺陷的定量原子场分析

A novel elemental and chemical analysis scheme based on electron-channeling phenomena in crystalline materials is introduced, where the incident ...

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a universally used traditional Chinese medicine) as an example. Ten batches of genuine Chuanmutong varieties and five batches of related adulterants were analyzed and compared based on the high-performance liquid chromatography (HPLC) fingerprints combined with chemometrics, including cluster analysis (CA), principal component analysis (PCA), and orthogonal partial least-squares discrimination analysis (OPLS-DA). In addition, the content of &#946;-sitosterol was determined. The control chemical fingerprint of Chuanmutong was established, and 12 common peaks were identified. The similarity between the fingerprint of 10 batches of genuine Chuanmutong varieties and the control fingerprint was 0.910-0.989, while the similarity of five batches of adulterants was only 0.133-0.720. Based on the common peaks in the chromatogram, 15 batches of samples were classified into three content levels by PCA, and were aggregated into four categories by CA, achieving a clear distinction between authentic Chuanmutong and adulterants of Chuanmutong. Further, seven differential components that can effectively identify authentic Chuanmutong and adulterants of Chuanmutong were found through OPLS-DA. The &#946;-sitosterol content of 10 batches of genuine Chuanmutong varieties was 97.53-161.56 &#181;g/g, while the &#946;-sitosterol content of the five batches of adulterants varied greatly, among which the &#946;-sitosterol content of Clematis peterae Hand.-Mazz. and Clematis gouriana Roxb. Var. finetii Rehd. et Wils. was significantly lower than that of authentic varieties of Chuanmutong. The HPLC index component content and chemical fingerprint multi-pattern recognition method established in this study provide a new strategy for effectively identifying authentic Chinese medicinal materials and related adulterants.

HPLC Coupled with Chemical Fingerprinting for Multi-Pattern Recognition for Identifying the Authenticity of Clematidis Armandii Caulis

Here, we present a protocol to establish high-performance liquid chromatography (HPLC), coupled with chemical fingerprint multi-pattern recognition, which provides a new strategy for effectively identifying the genuine varieties of Clematidis Armandii Caulis and its adulterants.

高效液相色谱结合化学指纹图谱进行多模式识别，鉴定金线莲真伪

A method for identifying Chinese medicinal materials and their related adulterants was constructed by taking Clematidis Armandii Caulis (Chuanmutong, a ...