Name: measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates
Uploaded: 2015-05-09T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

我们承认线虫遗传学中心C.线虫株。资金支持是由特里·福克斯研究院提供。我们也承认从罗朗德和马塞尔戈塞尔林研究生助学金，并在麦吉尔综合癌症研究训练计划的癌症研究FRN53888的CIHR / FRSQ培训补助授予EP支持。

1.准备试剂<ol><li>为C.制备的媒体线虫的生长（在这种情况下，野生型动物和FLCN-1（ok975）突变体动物）。 <ol><li>制备改性含5.5克的Tris盐酸2.4克Tris碱的，31克Bactopetone，20克氯化钠和0.08克胆甾醇的Youngren的只有细菌用蛋白胨（智管）干混。摇动拌匀。 注:这样的搭配足以准备10升MYOB介质。 注:正常生长培养基（NGM）板可用于智管板27代替。然而，应该用于菌株之间和独立重复之间有效的比较相同类型的板。在这个协议中，我们只用了MYOB板。 </li><li>制备1升智管培养基中加入6克智管干混17克琼脂和补至1L用H 2 O和高压釜中在122℃下45分钟。倒板，让干在室温2天。 </li><li>使用无菌技术，接种百毫升文化前进的再与E. LB肉汤培养基大肠杆菌 OP50细菌和长出O / N在37℃。 </li><li> MYOB种子板的E.大肠杆菌 OP50细菌。允许它生长在室温48小时。 </li></ol></li><li>利用RNA干扰基因下调准备<ol><li> MYOB准备RNA干扰喂养板。请按照步骤1.1.1和1.1.2。高压灭菌后，允许该介质冷却至约55℃并加入1 ml的1M异丙基β-D-1-硫代半乳糖苷（IPTG）和1ml 100mM的氨苄青霉素。 </li><li> E.连胜大肠杆菌 HT115细菌的质粒转化，其表达或者控制的EV或FLCN-1特异性RNAi对氨苄青霉素（50微克/毫升）/四环素（15微克/毫升）的琼脂板上。成长的文化O / N在37℃。 </li><li>挑选菌落，接种E.大肠杆菌 HT115细菌的质粒转化其表达或者控制的EV或FLCN-1特异性RNAi和在LB /氨苄青霉素（50微克/毫升）中，O / N在37＃生长培养176℃。 </li><li>种子板块与E.大肠杆菌 HT115 EV细菌或HT115 FLCN-1 RNA干扰细菌。 注意:保持板48小时，在RT使用前。如果RNAi敲低被馈送是在有效，使用递送的双链RNA的28的其他方法。 </li></ol></li><li>使用诱导百草枯（PQ）氧化应激: <ol><li>制备M9的缓冲区通过混合的KH 2 3克PO 4，6克的Na 2 HPO 4，5克氯化钠和0.25g硫酸镁4·7 H 2 O调出到1L蒸馏水。为在122℃和存储瓶在室温45分钟的高压釜溶液。 </li><li>在测定当天，制备含PQ的M9 /溶液。对于这个协议，使用100毫米PQ（0.1028克PQ溶于4毫升M9缓冲填满整个96孔板40微升每孔M9 / PQ的）。 注意:PQ是一个危险和剧毒化合物。根据适当的准则处理。 注:当第一次运行新的S火车或一个新的条件，建议测定生存PQ增加浓度来确定，以便在7-10小时杀死动物，用最好的浓度。 </li></ol></li></ol> 2.准备工作年龄同步L4人口<ol><li>转让20妊娠成人雌雄同体到一个新的MYOB板接种E.大肠杆菌 OP50细菌。备基于所述测定期间所需要的蠕虫数目几个平板。 </li><li>让他们产卵6小时，然后使用铂丝挑虫取出母亲。检查在显微镜下将板以评估产卵的数量。 </li><li>让鸡蛋孵化，成长在20℃48小时。此时，将动物处于晚期L4 /年轻的成年阶段。挑同一阶段的动物和转移到PQ含有溶液的孔中。 注意:由于48小时温育仅适用于生长类似于野生型动物的突变体，其监视与发育是重要新测试的突变体的速率，以确保它符合野生型发育率，否则，其它时间轴应使用。 </li><li>另外，使用同步技术。生成的使用次氯酸盐溶液的L1幼虫蠕虫同步人口（25毫升水中125毫升5.25％的次氯酸钠，加入50ml 4M氢氧化钠;包裹瓶用铝箔避光隔离）。然而，对于该测定，它是不推荐的，因为强烈的漂白可能影响测定期间的动物的行为。 注意:根据指引处理次氯酸钠溶液。 注:使用RNA干扰下调目的基因，如第2节，除了母亲转移到播种的特异性RNAi细菌RNA干扰板同步雌雄同体。当与RNA干扰击倒较弱，建议饲养多代。 </li></ol> 3.执行氧化胁迫的抗性测试&lt;/ P&gt; <ol><li>吸管40微升100mM的PQ溶液（新鲜制备），以96孔板的每孔的。使用至少12口井为每复制条件（治疗，突变等 ）。使用铂丝虫拾取，转移5-8 L4幼虫动物每孔指示的开始时间和结束时间。 </li><li>当开始另一个条件，请注意的开始时间和结束时间。将板在20℃在一小时后从开始时间传送所述蠕虫和得分死动物之间的温育时间。 </li><li>使用解剖显微镜，算上每生存小时。在开始之前，计数轻轻摇动板。表明蠕虫不动，甚至看上一个高强度的光后，为死去的动物。也表明动物存活的数量。 注:纵观蠕虫状，尾巴和头部运动在高放大倍率，帮助确定由活着死虫。 </li><li>直到大部分的蠕虫都死了每小时重复此步骤。 </li></ol> &lt;P类="j​​ove_title"&gt; 4。在各时间点测定存活率<ol><li>通过添加传送到每一个井动物的总数计算出每条件的动物的总数。忽略损坏或转让时被杀的动物。 </li><li>对于每一个时间点，计算死亡的动物的每种条件的总数（死的动物中的12个孔和）。对于每一个时间点上，计算死亡的百分比和存活（100减去百分之死亡）百分比（死亡动物100通过动物的总数除以并乘以数）。 </li><li>重复该测定的至少3个独立的倍。 </li></ol>

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S., Curtis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMP-activated+protein+kinase+AAK-2+links+energy+levels+and+insulin-like+signals+to+lifespan+in+C.+elegans.">The AMP-activated protein kinase AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans.</a> Genes Dev. 18, 3004-3009 (2004).</li><li>Lee, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+AMP-activated+protein+kinase+AAK-2+is+phosphorylated+by+LKB1+and+is+required+for+resistance+to+oxidative+stress+and+for+normal+motility+and+foraging+behavior.">The Caenorhabditis elegans AMP-activated protein kinase AAK-2 is phosphorylated by LKB1 and is required for resistance to oxidative stress and for normal motility and foraging behavior.</a> J Biol Chem. 283, 14988-14993 (2008).</li><li>Honda, Y., Honda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+daf-2+gene+network+for+longevity+regulates+oxidative+stress+resistance+and+Mn-superoxide+dismutase+gene+expression+in+Caenorhabditis+elegans.">The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.</a> FASEB J. 13, 1385-1393 (1999).</li><li>Restif, C., Metaxas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+swimming+motions+of+C.+elegans+worms+with+applications+in+aging+studies.">Tracking the swimming motions of C. elegans worms with applications in aging studies.</a> Med Image Comput Comput Assist Interv. 11, 35-42 (2008).</li><li>Buckingham, S. D., Sattelle, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast,+automated+measurement+of+nematode+swimming+(thrashing)+without+morphometry.">Fast, automated measurement of nematode swimming (thrashing) without morphometry.</a> BMC Neurosci. 10, 84 (2009).</li></ol>

The authors declare that they have no competing financial interests.

线虫是一个有吸引力的模式生物研究体内基因氧化应激抗性，因为它可以很容易地进行培养，并迅速导致大量基因完全相同的后代。多种方法来测量氧化应激抗性先前已经描述和它们是基于培养板与各种活性氧源如PQ，鱼藤酮，过氧化氢 ​​，和胡桃醌25,26,29-32的补充。在这里，我们描述了一种协议，用于测量氧化应激抗性在液体中使用100mM的PQ 96孔板中。类似的试验已被格里尔等 33进行。该测定可以进一步适于使用其他ROS源来研究氧化应激的液体。举例来说，我们已经表明了FLCN-1增加阻力损失数H 2 O 2的浓度以及23。 此法可能是技术立法院对于显示运动缺陷或某些菌株具有挑战性的游泳诱发瘫痪的表型，如株携带突变的多巴胺转运DAT-1 34。在这种情况下，无法移动是混乱的，因为它并不一定意味着减少氧化应激抗性而是运动缺陷。此外，动物尸体计数应认真完成，研究人员要注意C.线虫的行为细节，如尾动作，头部动作，甚至是缓慢的肢体动作。如果PQ浓度太高，和动物的死亡动力学是非常快的，人们可能会考虑在为了得到较慢的死亡率降低PQ的浓度。有些动物对氧化应激极为敏感，如AAK-2突变体动物26,33,35,36而有些则是高度耐药，如DAF-2突变体动物37。在这种情况下，测定存活具有不同共PQ的ncentrations应予以考虑。 为RNAi实验，阴性结果可能是由于所述RNAi治疗的低效率。在这种情况下，mRNA水平的测量是必要的，以确定拦截的基因是否是成功的。 使用一个探测器跟踪C的游泳行为，该协议可以进一步适应氧化应激性高通量筛选线虫液体38,39。这将潜在地使C的耐力监测线虫和游泳池的行为，如身体氧化压力下弯曲的频率。身体运动的更高的速率可能表明在压力下更高的蠕虫病毒健身。 通路，导致氧化低等真核生物胁迫抗性是高度保守的跨演变和链接到氧化应激相关的疾病和人类中老化。 Mitohormesis和平衡generatioñ活性氧是必不可少的延长寿命和提高健康水平跨度。然而，过度的活性氧是严重损害细胞，组织/器官和生物体。发现的途径，如果改变，提供最佳的活性氧平衡因此，必须和调节对氧化应激的抗性的屏幕对于药物可帮助固化的发展为多种疾病与公分母:氧化应激。执行这些场次中C.线虫是具有很大的潜力和价值的理解和处理挂钩氧化应激的人类疾病的一个有利的快速，廉价，可靠的方法。

A reference was corrected in the publication of <a href="http://www.jove.com/video/52746">Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates</a>. There was an error with reference 34 in the Citations section. It has been updated from:
Allen, E., Walters, I.B., Hanahan, D. Brivanib a dual FGF/VEGF inhibitor,is active both first and second line againstmouse pancreatic neuroendocrine tumors developing adaptive/evasive resistance toVEGF inhibition. Clin Cancer Res. 17, (16) 5299-310 (2011).
to:
Allen, A. T., Maher, K. N., Wani, K. A., Betts, K. E., &#38; Chase, D. L. Coexpressed D1- and D2-like dopamine receptors antagonistically modulate acetylcholine release in Caenorhabditis elegans. Genetics. 188, 579-590 (2011).

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

在真核生物中，氧化磷酸化发生在线粒体的电子传递链是能源生产中ATP形式的主要驱动力。活性氧（ROS）是这一进程的一个天然副产品。尽管它们作为信号分子的重要作用，过量的ROS可导致DNA损伤，蛋白质的羰基化和脂质氧化。 ROS产生和解毒之间的不平衡引起的氧化应激，从而导致能量消耗，细胞损伤，并触发细胞死亡1,2。氧化应激促成老化和许多威胁生命的疾病，包括癌症，糖尿病，心血管疾病和神经变性疾病3-9的发展。 细胞进化的酶和非酶防御战略，以保持适当的ROS水平，并保护他们的选民免受氧化损伤1,2。超氧化物歧化酶（SOD）酶首先采取行动，便利着想室温超氧成H 2 O 2，这将在后面转化为水通过过氧化氢 ​​酶或过氧化物酶。非酶促防御策略主要包括反应的速度与ROS相比，细胞大分子，保护重要的细胞成分的分子。尽管ROS解毒酶的保护作用，一些活性氧分子逃避抗氧化防御机制，导致氧化损伤。检测，修理，和受损的细胞成分的降解是氧化应激1,2-期间必需防御策略。 信号参与抗逆性和具体氧化应激的途径是高度进化保守10,11。与细胞培养实验中有机体的条件只有部分转载，氧化应激在模式生物12,13具有重要的意义的研究。C.线虫是一种自由生活的线虫，可以容易且廉价地尽头捕获的原始图像上的菌苔上的琼脂培养基。它的尺寸小（约1毫米长），并通常生长作为自我施肥雌雄同体，这有利于遗传操作。它有一个快速的生命周期和较高的繁殖能力，生产每代300的后代，使之成为一个强大的工具来执行大规模的遗传筛选14。 C.该线虫基因组被完全测序和基因的40-50％被预测为人类疾病相关的基因15-18的同系物。利用RNA干扰目的基因的敲除快速，容易C.线虫。基因下调可通过饲养动物的大肠杆菌可以实现杆菌细菌窝藏表达靶向感兴趣19的mRNA表达的双链RNA的质粒。因此，确定采用大规模RNAi筛选基因的功能，对了解人类疾病，包括癌症20,21很大的影响。 邻研究在C xidative抗逆性线虫已经导致电阻的保守机制的鉴定对氧化应激13,22。确定了一些途径是调节长寿和耐其它应力以及如缺氧，热和渗透胁迫共同通路。这些途径包括胰岛素信号，TOR信令，和自噬。其他主要途径包括ROS的解毒，如超氧化物歧化酶酶和过氧化氢 ​​酶，或损伤修复，如热休克和伴侣蛋白11,13,22。 这个协议描述了如何确定到C的氧化应激的抗性线虫在液体中。我们使用FLCN-1（ok975）和野生型动物表现出的协议，因为我们先前在C时的损失示于氧化应激的抗性增加FLCN-1（ok975）线虫 23。我们还表明，这种增加的电阻取决于对AMPK和自噬，信令轴，提高了细胞生物能量，促进抗逆性23。 PQ是氧化应激，与电子传递链，以产生活性氧24干扰。相同的测定可以适于和其他活性氧源或活性氧生成的化合物可以使用如H 2 O 2和鱼藤酮。类似测定法已开发上，其中低浓度PQ的使用25,26板。该测定的优点在于，它是非常快，其结果可能会在一天内获得。此外，液用于在96孔板进行氧化应激抗性测试的总体积是低相比，用于制备含PQ板的体积。因此，PQ的用量是在液体中测定低，这使得该测定便宜并限制生产有毒废料。然而，该测定的相比，板测定法限制包括拉食物中的液体化验和氧气在液体中的低浓度的放相比于空气。这些是在某些情况下，可能会影响结果的重要因素。因此，在使用的氧化应激抗性的其它方法，推荐以支持在该测定中获得的结果，确认再现性。

<table><tbody><tr><td>Agar bacteriological grade</td><td>Multicell</td><td>800-010-LG</td><td></td></tr><tr><td>Bacteriological peptone</td><td>Oxoid</td><td>LP0037</td><td></td></tr><tr><td>Sodium chloride biotechnology grade</td><td>Bioshop</td><td>7647-14-5</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8503-25G</td><td></td></tr><tr><td>UltraPure tris hydrochloride</td><td>Invitrogen</td><td>15506-017</td><td></td></tr><tr><td>Tris aminomethane</td><td>Bio Basic Canada Inc</td><td>77-86-1</td><td></td></tr><tr><td>IPTG</td><td>Santa Cruz Biotechnology</td><td>sc-202185A</td><td></td></tr><tr><td>Ampicillin</td><td>Bioshop</td><td>69-52-3</td><td></td></tr><tr><td>Yeast extract</td><td>Bio Basic Inc.</td><td>8013-01-2</td><td></td></tr><tr><td>Methyl viologen dichloride hydrate</td><td>Aldrich chemistry</td><td>856177-1G</td><td></td></tr><tr><td>Petri dish 60 x15 mm</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Pipet 10 ml</td><td>Fisher</td><td>1367520</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>G-Biosciences</td><td>RC-084</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M-5921</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Bioshop</td><td>7558-79-4</td><td></td></tr><tr><td>Discovery v8 stereo zeiss microscope</td><td></td><td></td><td></td></tr><tr><td>96 well clear microtiter plate</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;flcn-1&lt;/em&gt; RNAi source</td><td>Ahringer Library</td><td></td><td></td></tr></tbody></table>

measuring oxidative stress resistance of caenorhabditis elegans in 96-well microtiter plates

氧化应激，这是生产和活性氧解毒之间的不平衡的结果，是一个主要贡献者慢性人类疾病，包括心血管和神经变性疾病，糖尿病，老化和癌症。因此，研究氧化应激不仅在细胞系统中，但也使用整个生物体是重要的。C.线虫是一个有吸引力的模式生物，研究了氧化应激信号转导通路，这是高度进化上保守的基因。 在这里，我们提供了一个协议来衡量C.氧化应激性线虫在液体中。简言之，将ROS诱导试剂如百草枯（PQ）和H 2 O 2溶解在M9缓冲液中，溶液等分在96孔微量滴定板的孔中。同步L4 /年轻的成年C.线虫动物被转移到孔中（5-8只动物/孔）和存活测定每隔一小时，直到大部分的蠕虫都死了。当使用在板的压力源的低浓度，老化可能会影响在氧化应激的动物，这可能导致不正确的数据的解释的行为进行了氧化应激抗性测试。然而，在本文描述的测定中，这个问题不太可能发生，因为只L4 /年轻的成年动物的使用情况。此外，该协议是廉价且结果在一天，这使得该技术对于遗传筛选吸引力获得。总的来说，这将有助于理解氧化应激信号传导途径，其可以转化成氧化应激相关的人类疾病更好表征。

比较野生型C.线虫到FLCN-1（ok975）突变体动物在这里，我们使用100毫PQ，以确定野生型C的电阻线虫相比FLCN-1（ok975）动物已显示抗氧化应激，热和缺氧23。 4小时的治疗，野生型的48.3％后存活相比77.8％的存活率在FLCN-1（ok975）动物。正如所料，FLCN-1（ok975）突变体动物是更耐至100mM PQ相比野生型（ 图2和表1）。 比较野生型动物喂以对照的EV或FLCN-1的RNAi菌类似的结果在上一节中介绍，向下FLCN-1的调节使用的RNAi增加了阻力至100mM PQ。该结果证明基因功能的使用的RNAi模拟米丧失功能的该下调<html> utation（ 图2和表1）。 <img alt="图1" src="/files/ftp_upload/52746/52746fig1.jpg" /> 氧化应激抗性的方法测定的在96孔板中在C图1原理图中线虫 。 同步生长在智管板和用大肠杆菌 OP50细菌送入L4 /年轻的成年动物被转移到含有100mM的PQ 96孔微量滴定板的孔中和存活测定每小时直到大量蠕虫都死了。在RNA干扰击倒的情况下，同样的步骤，只是该同步动物生长在补充有IPTG平板，和被馈送以大肠杆菌大肠杆菌 HT115细菌窝藏，将在表达时拦截靶基因的质粒。 文件/ ftp_upload / 52746 / 52746fig2.jpg"/&gt; 图2.损失FLCN-1的增加的抗氧化应激的C.线虫 （A - B）的百分比存活至100mM的PQ（A）的重量和FLCN-1（ok975）突变体动物和（B）的重量与对照治疗或FLCN-1的RNAi动物。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td colspan="5"> 生存的百分比为100毫米PQ </td></tr><tr><td> 1小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 82.38±1.31 </td><td align="right"> 0.0002 </td><td align="right"> 3 </td><td align="right"> 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </td><td> 99.03±1.67 </td><td></td><td align="right"> 3 </td&gt; <td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 91.70±0.55 </td><td align="right"> 0.0462 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 95.93±2.48 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 2小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 72.13±7.13 </td><td align="right"> 0.005 </td><td align="right"> 3 </td><td align="right"> 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </td><td> 96.33±2.28 </td><td></td><td align="right"> 3 </td><td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 80.27±5.34 </td><td align="right"> 0.0261 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 91.23±1.42 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 3小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 55.37±4.72 </td><td align="right"> 0.0004 </td><td align="right"> 3 </td><td align="right"> 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </eM&gt; </td><td> 87.00±1.36 </td><td></td><td align="right"> 3 </td><td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 70.77±3.50 </td><td align="right"> 0.0027 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 87.63±2.67 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 4小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 48.30±6.39 </td><td align="right"> 0.002 </td><td align="right"> 3 </td><td align="right"> 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </td><td> 77.80±3.12 </td><td></td><td align="right"> 3 </td><td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 63.77±0.66 </td><td align="right"> 0.0122 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 80.57±6.68 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 5小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 41.60±8.33 </td><td align="right"> 0.0062 </td><td align="right"> 3 </td><td a河旁="右"&gt; 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </td><td> 67.43±1.42 </td><td></td><td align="right"> 3 </td><td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 60.53±2.58 </td><td align="right"> 0.0313 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 71.53±5.24 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 6小时</td><td>应变，RNA干扰</td><td>百分比生存（±SD） </td><td>中P值</td><td>实验数</td><td>蠕虫总数</td></tr><tr><td></td><td>重量</td><td> 34.86±5.88 </td><td align="right"> 0.0021 </tD&gt; <td align="right"> 3 </td><td align="right"> 210 </td></tr><tr><td></td><td> FLCN-1（ok975） </td><td> 60.34±2.05 </td><td></td><td align="right"> 3 </td><td align="right"> 224 </td></tr><tr><td></td><td>重量;控制</td><td> 44.43±3.93 </td><td align="right"> 0.0042 </td><td align="right"> 3 </td><td align="right"> 202 </td></tr><tr><td></td><td>重量; FLCN-1（RNAi）技术</td><td> 62.13±3.44 </td><td></td><td align="right"> 3 </td><td align="right"> 207 </td></tr></tbody></table> 表1的结果和从图2中的氧化应激抗性的统计分析的综述。

Watch this Scientific Journal Video about Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates at JoVE.com

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-oxidative-stress-resistance-caenorhabditis-elegans-96-well

Nanyang Technological University

Research

JoVE Journal

Biology

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

15.4K 次观看.  McGill University. 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. 

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

遗传学

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

线虫中耳炎群链球菌引起氧化应激的研究

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

发育生物学

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

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 ...

生物学

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

线虫的耗氧率测定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://www.jove.com/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

秀丽隐杆线虫存活试验:短期毒性试验

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

测量氧化应激性

摘要

探索更多视频

此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

线虫中耳炎群链球菌引起氧化应激的研究

测量线虫寿命在96孔板

线虫的耗氧率测定

C. Elegans生理应激反应的测量

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响

秀丽隐杆线虫存活试验:短期毒性试验

测量氧化应激性

摘要

探索更多视频

此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

调查低成本方法，以测量秀丽隐杆线虫的寿命和健康寿命

线虫中耳炎群链球菌引起氧化应激的研究

测量线虫寿命在96孔板

线虫的耗氧率测定

C. Elegans生理应激反应的测量

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

微生物分离株的高通量筛选对 秀丽隐杆线虫 健康的影响

秀丽隐杆线虫存活试验:短期毒性试验

微生物分离株的高通量筛选对秀丽隐杆线虫健康的影响