这个协议最初是由小榄镇叶和迈克尔Petrascheck琳达巴克实验室在西雅图的弗雷德哈钦森癌症研究中心。上面详细的版本已经准备提供更广泛的社会，使整个过程。我们感谢卡罗尔泰勒，萨拉LeBoeuf和安迪Tomacelli批判地阅读协议。这是来自斯克里普斯研究所的手稿＃2866。 Petrascheck实验室是由诺华ADI方案。

概述:该协议是分割成四个部分。第1部分介绍了如何准备喂食细菌。第2部分介绍了如何编写的蠕虫文化。第3部分介绍了如何得分寿命。第4部分展示了一些有代表性的结果，第5部分介绍了如何准备所需的解决方案。寿命实验需要几个星期才能完成。协议一周的每一步，包括一天，一天的时间，以方便规划。作为整个协议的参考点的L4阶段（0天）。 1。饲喂细菌的制备本节介绍了准备喂食细菌。具体的大肠杆菌菌株用于饲料线虫被称为OP50。在此协议，与其他细菌的蠕虫文化，以防止交叉污染，已取得OP50应变羧/氨苄西林耐药 21 。预先准备的OP50四到五天。在接触与OP50的所有材料必须是无菌的。 日-7日（星期四）（1周）:接种5毫升一个OP50殖民地，并培育在夜间在37℃一个细菌摇床中的C与含有100微克/ mL氨苄青霉素和0.1微克/毫升两性霉素B的结核病。 日-6日（星期五）（1周）。 上午8:30。早接种，以便有足够的时间为文化达到饱和<ol><li>在300毫升含50μg/ mL氨苄青霉素的结核病OP50 1:2000稀释过夜培养。为8-12小时，细菌在37℃摇床孵育文化，直至达到饱和。不要让文化不再增长超过14小时。 </li><li>转移到无菌的，预加权的离心管OP50。沉淀离心10分钟在3500转的离心机表（2200 XG）OP50。 </li><li>弃上清，重新悬浮在无菌水OP50颗粒和颗粒重新离心。重复洗两次。 </li><li>第二次洗涤后，小心地取出所有剩余的水。没有水，应留在管。称取离心管中的颗粒和减去空离心管的重量，以确定沉淀的重量。 </li><li>重新彻底停止在S -完整的颗粒浓度为100毫克/毫升。应留给无团块。 </li><li> 100毫克/毫升OP50的浓度应与2 × 10 10个细菌/ mL的。使用照片的光谱仪来确定每毫升的细菌数量，如果每毫升细菌之间的光密度和数量的关系是已知的。如果有必要，调整OP50喂养的解决方案，以2 × 10 10个细菌/ mL的浓度。 </li><li>商店OP50solution在4 ° C直到蠕虫文化。 </li></ol> 2。同步蜗轮文化的制备本节介绍了编写的蠕虫文化。它的目标是产生一个蠕虫的人口年龄同步。 线虫在接触后，在第5步中的漂白剂治疗的所有材料必须是无菌的。板都保持在20 ° C，除非另有说明。 -6日:周五（1周），下午4:00: 动物转移到一个新的板块 <ol><li>以5-10日龄NGM的板大多数蠕虫人口饿死L1幼虫。 </li><li>不久超过本生灯加热消毒金属锅铲。让它冷却。使用冷却铲切出琼脂块板饿死蠕虫。转移到一个新的10厘米NGM板种子与OP50这几大块。孵育约。 65小时在20℃直至蠕虫人口的大多数妊娠的成年人组成。为饿死的L1动物成长为妊娠成人的时间可能会有所不同应变应变。 </li></ol> 日-3:周一（2周）上午10:00: 建立同步的人口<ol start="3"><li>从10厘米的板收集洗涤他们用5-10毫升无菌水盘蠕虫。转移到15 mL锥形管蠕虫/水溶液。 </li><li>清洗离心2分钟，在台式离心机1200 RPM（280 XG）的蠕虫病毒。弃上清，加入10 mL水。重复。 </li><li>去除上清液，加入5毫升的新鲜配制的漂白剂/氢氧化钠溶液（2.5毫升的家用漂白剂，1毫升10N氢氧化钠，6.5 mL的H 2 O） 。为5分钟，在室温下孵育直到蠕虫破开。务必旋涡轻轻每分钟。监视解剖显微镜下的反应的进展。 </li><li>只要所有的成年人溶解，加5毫升M9的缓冲液中的反应。 </li><li>鸡蛋洗净用10 mL M9的缓冲液，离心2分钟2500转三次（1100 XG）。 </li><li>一旦与洗净的鸡蛋，10毫升的S - 2500 RPM（1100 XG）离心2分钟完成。 </li><li>去除上清，加入10 ml的S -完整的解决方案转移到一个新的50 mL锥形管。新增的S完成30毫升到40毫升的最终体积。在室温下轻轻摇动管nutator或类似设备上过夜。 </li></ol> -2日:星期二（2周），中午12:00: 种子的动物进入板 <ol start="10"><li>根据解剖范围，检查是否蠕虫在夜间孵化。蠕虫计数的蠕虫数量在10μL，滴，用解剖范围的S -完整的解决方案中确定的浓度。计数每个样品至少10滴。 </li><li>重新暂停集中在80-100蠕虫/毫升的S -完全的蠕虫。添加羧（库存100毫克/毫升）至终浓度为50μg/ mL和两性霉素B（库存250微克/毫升）的一个终浓度为0.1微克/毫升。摇上一个nutator。如果编写的蠕虫病毒数量较多，使用过滤器的第一个600毫升nunclon烧瓶。 </li><li>下午2时30分添加到终浓度为6毫克/毫升（= 1.2 × 10 9菌/毫升）在第1部分准备OP50。返回OP50到4 ° C。 </li><li>传送到每一个96孔板以及120μL的worm/OP50溶液。使用96孔板，用透明的底部。请务必保持悬浮液中的蠕虫，而吹打。 </li><li>密封板，使用胶带封口机，以避免污染和蒸发。摇晃2分钟，在微孔板摇床板，2天在20℃孵育，直到动物到达的L4阶段。 </li></ol> 0天:星期四中午十二时前（2周）: 加入Fluorodeoxyuridine消毒动物（FUDR） <ol start="15"><li>消毒的动物添加30μL0.6毫米FUDR原液，以每口井。这一步，使每孔终体积为150μL和减少6mg /毫升的OP50终浓度为5 mg / mL的菌（1 × 10 9 /毫升） 。用胶带封口机，封板，握在酶标板振动筛为2-3分钟。如果OP50被添加在下午2时30分-2天，重要的是，FUDR是中午前添加。 20℃培养箱返回板块</li></ol> 第1天:周五（2周）: 添加药物文化 <ol start="16"><li>上午9:00动物最应该含有妊娠和几个鸡蛋。新增的对寿命的影响是在测试所需浓度的药物。如果药物溶解在DMSO，DMSO的终浓度应不超过0.6％，为二甲基亚砜浓度超过0.6％，缩短线虫寿命。另外的药物后，用胶带封口机密封板和动摇，在酶标板振动筛为2-3分钟。 20℃培养箱返回板块</li><li>添加的药物，偶尔可以杀死每盘几个动物，特别是如果使用其他溶剂比水。使用倒置显微镜检查动物尸体。一般来说，应低于10％，96孔板中死亡的动物。返回板块到20℃培养箱。 </li></ol> 4日:周一（3周）: 更改封口机 <ol start="18"><li>为了让新鲜的氧气进入文化删除封口机，等待1分钟放回板。摇板为2-3分钟，在微孔板摇床。重复每星期一次。 </li></ol> 第5天:周二（3周）: 添加新的OP50，以防止饥饿<ol start="19"><li>添加5μL的100毫克/毫升OP50的解决方案，准备在第1部分的96口井，以防止饥饿。 </li></ol> 3。评分的寿命。 本节介绍如何生存的第2部分同步蠕虫人口监测，直到动物死亡。要观察的动物在96孔板，用倒置显微镜的2倍或2.5倍的目标。生存记录数据，每周三次，周一，周三和周五。使用运动，以确定是否是活着还是死了的动物。强光照射，尤其是蓝色的光芒，诱使动物移动。不要从实验中删除​​的死动物。偶尔，不动，并决心在前面的计数死的动物可能会稍后动议。 <ol><li> 0天，在实验开始计数，每孔总数的蠕虫。审查井包含超过18动物从分析这些水井中的动物不会有足够的OP50，将显示限制饮食的影响。 </li><li>为了增加机会，动物移动撼动96孔板上微孔板摇床2分钟。计数前。 </li><li>在每一个计数，记录会议的日期和动物数量移动作为活的动物。在液体中的运动是比固体培养基上容易得多，并可以由强光引起的。一个更高的放大倍率的使用可能有助于发现咽的一角非常细微的动作。这些细微的动作往往只有在非常古老的动物运动观察。 </li><li> 20℃培养箱返回板块</li><li>重复步骤2-4，每两至三天，直到所有的动物都死了。 </li></ol> 4。代表性的成果。 本节说明如何保持这个实验和一些有代表性的结果所产生的寿命数据记录的一个例子。 图1A显示了如何在这个实验中的寿命数据记录的一个例子。一个Excel工作表是用来保持在每口井的人口的生存轨道。每口井的坐标中板，应变，药物，药物浓度和0天，活着的动物总数（x0）是在实验开始记录。记录日期，以及每周三次以按照每口井的不同人群的生存生活和死亡的动物的数量。图中的结果，计算出每一天活着的动物的一小部分和情节，在几天的时间功能。应使用Stata或类似的软件统计软件包计算P值。 线虫中等寿命是取决于温度。图1b显示，寿命取决于温度的变化，准确再现基于微孔板的寿命检测22。同样，微孔板检测再现据报道，寿命从野生型N2动物23-24 25（图1C）不同的突变体寿命的变化。 该试剂盒还可以被用来制造更定量的陈述。 Fig1D平均寿命是绘制Mirtazepine浓度7功能。每个数据点代表7-12人口的平均寿命，每个生活在不同的井。即使每口井的动物的数量是比较低的（5-15动物）的的井 - 井的变化相对较小，因为可以从错误酒吧。 <img src="/files/ftp_upload/2496/2496fig1.jpg" alt="图1" /> 图1:基于微孔板的寿命检测，可精确地再现寿命的变化文献报道。 （一）样本收集的数据在Excel电子表格。在每个计数会议的日期，协调以及录得活的和死的动物的数量。在实验开始时，每口井的动物总数确定。 （二）生存的野生型曲线（N2）动物，生长在20 ° C和25 ° C。 （三）携带株​​基因突变，影响寿命的生存曲线。所有4株在20 ° C平行测定（四）Mirtazepine治疗氮气动物的剂量响应曲线。平均寿命是绘制Mirtazepine浓度的功能。误差棒表示教统局局长的8％条件井。 5。材料。 M9的缓冲区，1000毫升<ul><li> 6克NA 2 HPO 4 </li><li> 3克KH 2 PO 4 </li><li> 5 g氯化钠</li><li> 0.25克硫酸镁4∙7H 2 O </li><li>加入去离子水1000毫升。 </li><li>高压灭菌器</li></ul> Potassiumphosphate缓冲液pH 6.0，1000毫升<ul><li> 136克KH 2 PO 4 </li><li>加入去离子水900毫升。 </li><li> 5M KOH调整pH值至6.0 </li><li>加入去离子水1000毫升。 </li><li>高压灭菌器</li></ul>微量金属解决方案<ul><li> 1.86克钠2 EDTA </li><li> 0.69克FESO 4∙7H 2 O </li><li> 0.20克MnCl 2∙4H 2 O </li><li> 0.29克硫酸锌4∙7H 2 O </li><li> 0.016克硫酸铜4 </li><li> 1000毫升去离子水</li><li>在黑暗中的高压灭菌器和存储。 </li></ul> S - 1000毫升培养基， <ul><li> 5.9 g氯化钠</li><li> 50毫升的1M磷酸钾，pH值6.0 </li><li> 1000毫升去离子水</li><li>高压灭菌器</li><li>让冷却解决方案，然后添加1毫克/毫升的胆固醇（溶解在ET - OH）毫升。 </li></ul>柠檬酸钾1M，1000毫升<ul><li> 268.8克三钾柠檬酸</li><li> 26.3柠檬酸一水柠檬酸</li><li>加入900毫升去离子水</li><li> 5M KOH调整pH值至6.0 </li><li>加入去离子水1000毫升。 </li><li>高压灭菌器</li></ul> S -完全培养基，1000毫升<ul><li> 977毫升的S -基底</li><li> 10毫升1M柠檬酸钾pH值6（无菌） </li><li> 10毫升的微量金属溶液（无菌） </li><li> 3毫升1M 氯化钙 （无菌） </li><li> 3毫升1M 硫酸镁 （无菌） </li></ul> NGM琼脂<ul><li> 3.0克氯化钠</li><li> 2.5克Pepton（从酪蛋白，胰腺消化） </li><li> 17克琼脂</li><li>加入去离子水975毫升，搅拌棒</li><li>高压灭菌器</li><li>经过高压灭菌降温至55 ° C和添加下列组件: <ul><li> 0.5毫升的1M 氯化钙 （无菌） </li><li> 1毫升5毫克/毫升的乙醇中的胆固醇</li><li> 1毫升的1M 硫酸镁 （无菌） </li><li> 25毫升Potassiumphosphate的缓冲液，pH值6.0（无菌） </li></ul></li></ul>结核病，1000毫升<ul><li> 12克细菌用胰蛋白胨</li><li> 24克酵母提取物</li><li> 4毫升甘油</li><li>加入900毫升去离子水</li><li>高压灭菌器</li><li>经过高压灭菌降温至55 ° C和添加以下组件: <ul><li>加入100毫升0.17M KH 2 PO 4 / 0.72MK 2 HPO 4 </li></ul></li></ul> 0.6毫米Fluorodeoxyuridine（FUDR，SIGMA猫＃F0503），1000毫升<ul><li> 100毫克FUDR </li><li>溶解在670毫升无菌的S -完备的，10毫升或45毫升分装。 </li><li>储存在-20 ° C。 </li></ul> 100毫克/毫升羧，10毫升<ul><li> 1克羧</li><li> 10毫升无菌去离子水</li><li>无菌滤液和等分</li><li>储存在-20 ° C。 </li><li>在终浓度为50微克/毫升的使用</li></ul> 250ug / mL的两性霉素B，4毫升<ul><li> 1mg的两性霉素B </li><li> 4毫升ET - OH </li><li>储存在-20 ° C </li><li>使用终浓度在0.1微克/毫升</li></ul>

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L., Benzer, S., Min, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+extension+in+Drosophila+by+feeding+a+drug.">Life extension in Drosophila by feeding a drug.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 838-843 (2002).</li><li>Avanesian, A., Khodayari, B., Felgner, J. S., Jafari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamotrigine+extends+lifespan+but+compromises+health+span+in+Drosophila+melanogaster.">Lamotrigine extends lifespan but compromises health span in Drosophila melanogaster.</a> Biogerontology. 11, 45-52 (2010).</li><li>Melov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+life-span+with+superoxide+dismutase/catalase+mimetics.">Extension of life-span with superoxide dismutase/catalase mimetics.</a> Science. 289, 1567-1569 (2000).</li><li>Benedetti, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+that+confer+thermal+stress+resistance+and+extended+lifespan.">Compounds that confer thermal stress resistance and extended lifespan.</a> Exp. Gerontol. 43, 882-891 (2008).</li><li>Braungart, E., Gerlach, M., Riederer, P., Baumeister, R., Hoener, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+MPP++model+of+Parkinson's+disease+for+high-throughput+drug+screenings.">Caenorhabditis elegans MPP+ model of Parkinson's disease for high-throughput drug screenings.</a> Neurodegener Dis. 1, 175-183 (2004).</li><li>Kwok, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+screen+in+C.+elegans+yields+a+new+calcium+channel+antagonist.">A small-molecule screen in C. elegans yields a new calcium channel antagonist.</a> Nature. 441, 91-95 (2006).</li><li>Moy, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screen+for+novel+antimicrobials+using+a+whole+animal+infection+model.">High-throughput screen for novel antimicrobials using a whole animal infection model.</a> ACS Chem Biol. 4, 527-533 (2009).</li><li>Kaletta, T., Hengartner, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+function+in+novel+targets:+C.+elegans+as+a+model+organism.">Finding function in novel targets: C. elegans as a model organism.</a> Nat Rev Drug Discov. 5, 387-398 (2006).</li><li>Chung, C. T., Niemela, S. L., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+preparation+of+competent+Escherichia+coli:+transformation+and+storage+of+bacterial+cells+in+the+same+solution.">One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.</a> Proc. Natl. Acad. Sci. U. S. A. 86, 2172-2175 (1989).</li><li>Klass, M. 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A. 96, 7394-7397 (1999).</li><li>Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+mutant+that+lives+twice+as+long+as+wild+type.">A C. elegans mutant that lives twice as long as wild type.</a> Nature. 366, 461-464 (1993).</li><li>Lakowski, B., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+life-span+in+Caenorhabditis+elegans+by+four+clock+genes.">Determination of life-span in Caenorhabditis elegans by four clock genes.</a> Science. 272, 1010-1013 (1996).</li></ol>

该协议提出让线虫寿命在96孔板的测量。代表性的结果显示部分，可靠地复制以前的研究结果，并提供了定量信息。使用这个实验中，我们已经成功地放映了89000分子， 他们对线虫寿命的影响。 药物筛选的目的，衡量一个96孔微孔板格式的寿命古典固体培养基检测的几个优点。它降低了媒体的准备，孵化空间的数量，以及所需的药量所需的劳动。 96格式和显微镜设置，使整个检测自动化的高通量放映。 多个值，每个变量和组合，在检测的发展及其对线虫寿命的影响进行了测试。这些测试包括不同OP50浓度范围从3至10毫克/毫升（3，4，6，8，10毫克/毫升），每口井的7至45（7，10，15，22，45中的蠕虫病毒的不同数量的蠕虫/孔），不同的文化卷从40到150μL，每孔（40，60，80，100，120，150μL），并在缓冲液成分的变化。如果美联储与羧耐OP50，既不羧也不两性霉素B的浓度表示，发现影响线虫寿命。连续轻轻摇动的板块，往往是线虫液体培养的建议，被发现在本实验中使用的小卷，缺一不可。然而，轻轻摇动，如果要使用较大的井微孔板。该值表示在这个协议中都经过精心挑选的基础统计对比测试条件的不同。 这个实验最令人惊奇的功能可能是线虫可保持在96孔板，用胶带密封。端侧比较没有透露任何启封板养殖动物的寿命的差异，在用塑料袋封口机密封板，或用封口机密封板，使空气的交换。在所有这三个条件，发达的动物非常均匀，从L1到65小时内妊娠成人，并表现出非常相媲美的寿命。上NGM的平行生长的动物发展速度稍快，但这种差异是独立的缺失或封口机的存在。 所提出的协议是基于活的细菌，但死菌可以适应。然而，在液体中检测单一幸存的细菌迅速繁殖并重新填充的文化。在我们的手中，唯一可靠的方式来杀灭细菌，它们可用于液体培养使用范围是由伽玛射线的细菌长期治疗。 寿命实验的规划考虑的一个重要的一点是检测电源。上的影响的大小而定，利息药物的效果，必须在多口井进行测试。 4药物治疗在一个典型的检测和4个控制井，大致对应40-50动物，应该足以检测在寿命增加30％，超过95％的实验。 14％的升幅只有60％的情况下，检测，因此需要更多的复制井。 丰富的寿命这个实验产生的数据可用于开发的实验或应变具体的参数化姜氏模型1。这些姜氏模型是有用的，以确定检测的权力和估计的大型屏幕的误报和底片。我们在盲目的实验验证这些模型的预测，并用他们的估计数字在大屏幕上的假阴性的（未发表结果）。 综上所述，我们预计，法将被大量使用，以确定小分子，延长线虫寿命和研究的基本机制。

A correction to the article Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates was made.

Erratum: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>cat# 171375</td>
<td>Also called Fungizone</td>
</tr>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>cat# F0503</td>
<td>FUDR is inactivated by heat, thaw in cold water</td>
</tr>
<tr>
<td>Mianserin</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>M2525-250MG</td>
<td>Use as positive control at 50&mu;M final concentration</td>
</tr>
<tr>
<td>Cyproheptadine</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>cat#C6022-MG</td>
<td>Alternative positive control at 10 &mu;M final concentration</td>
</tr>
<tr>
<td>Sealing Tape</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>cat# 236370</td>
<td>Polyester, non-sterile</td>
</tr>
<tr>
<td>96 well plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>cat# 351172</td>
<td>Non-treated, transparent, sterile individual packaged, polystyrene</td>
</tr>
<tr>
<td>Nunclon flask</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>cat# 178883</td>
<td>used for large volumes</td>
</tr>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span in 96 well microtiter plates

寿命几个遗传途径的生物过程的监管。调查老化生物学的策略之一是研究动物的年龄监管途径的组成部分海港突变。如果这些基因突变的扰动的年龄调控通路的功能，因此改变了整个生物体的寿命，它们提供了重要的机械见解1-3。 另一种策略，以探讨规管的寿命是使用小分子扰乱年龄调控途径。到目前为止，分子数是已知的各种模式生物的寿命延长，并作为工具，用于研究生物老化4-16。因此，确定分子的数量远远相比是很小的遗传"工具集"，是研究衰老的生物学的。 线虫是用于研究，因为其出色的遗传学和短寿命的三个星期老化的原则模型之一。最近， 线虫作为模式生物表型为基础的药物屏幕5,7,16-20由于其体积小，其成长能力微孔板出现。 在这里，我们提出了一个实验来衡量线虫寿命在96孔板。该试剂盒的开发，并成功地用于屏幕延长线虫寿命 7的分子的大型图书馆。在多个测试，检测的可靠性进行了评估:第一，通过测量在不同温度下生长的野生型动物的寿命;第二，通过测量与改变的寿命突变体的寿命;第三，通过测量不同浓度的寿命变化的响应抗抑郁药Mirtazepine。 Mirtazepine先前已被证明寿命延长线虫 7。这些测试的结果表明，该检测是能够复制以前的研究结果从其他实验和定量。微孔板格式，也使得这个寿命检测与自动化液体处理系统兼容，并允许将集成自动化平台。

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates at JoVE.com

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

在这个协议中，我们提出了一个方法来衡量<em&gt;线虫</em在96孔板的使用寿命。

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

measuring-caenorhabditis-elegans-life-span-96-well-microtiter

Nanyang Technological University

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

36.4K 次观看.  The Scripps Research Institute. 在这个协议中，我们提出了一个方法来衡量线虫

视频: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

科研

生物学

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

量化老化细胞生长的酵母年谱跨度

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

时间推移显微镜早期胚胎发育线虫

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

冰帽:为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法:同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

前列腺素的提取与分析<em&gt;线虫</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

测量线虫寿命在96孔板

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测量线虫固体介质的寿命

量化老化细胞生长的酵母年谱跨度

在芽殖酵母中的自我复制寿命测量

时间推移显微镜早期胚胎发育线虫

冰帽:为发展方法拟南芥和番茄植株在96孔板高通量的基因分型

基本的

前列腺素的提取与分析

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

测量氧化应激性

测量在C.elegan的RAN肽毒性