这项工作是支持老年学奖和美国国立卫生研究院资助1R01AG031108 - 01 MKGS是由美国国立卫生研究院培训资助P30AG013280支持格伦/ AFAR突破。 Mk为中老年埃利森医学基金会新学者。

第1部分：准备线虫生长介质（NGM）板本节介绍了在寿命实验中使用的固体NGM板的准备。一个基本的寿命实验需要两种类型的板块：NGM的标准板，它含有任何添加剂，并安培/ FUDR板，氨苄青霉素（AMP）和fluorodeoxyuridine（FUDR）添加到了NGM。氨苄青霉素是用来防止外来细菌污染。 FUDR抑制细胞分裂，降低产蛋量，防止种蛋。 FUDR为长寿分析使用，不影响成人的寿命，不再需要转移蠕虫每隔几天以他们分开增长幼虫。板块的这两种类型的种子与E.大肠杆菌 OP50细菌，随后暴露在紫外线中丧生。 <ol><li>准备NGM（见配方和存储说明第5条）和标签的培养皿板。您将需要1 60毫米，每10毫升的NGM的培养皿板。如果你是与先前准备的坚实NGM开始，继续到步骤1.2。如果你是新鲜的蒸压NGM的开始，请跳到步骤1.4。 </li><li>在30个脉冲微波高，完全融坚实的NGM的。脉冲之间的漩涡媒体建设，以防止压气泡。 </li><li>将液体NGM在55℃水浴或冷却在板凳上。 </li><li>一旦NGM达到55 ° C添加33μL的150毫米FUDR和100μL100毫克/ mL氨苄青霉素，每100毫升NGM（安培/ FUDR板）和漩涡混合。无需额外的药物NGM板，直接进行步骤1.5。 </li><li>使用移液器10毫升NGM的无菌技术，每60毫米培养皿板。避免形成气泡如果可能的话。如果气泡的形式，他们可以使用枪头弹出。 </li><li>在板凳上保留与上眼睑板允许NGM的巩固和干。如果可能的话，离开板凳上板2天前加入细菌，但如果需要越早板，1天工作。 </li><li>当天板块之前完成从新鲜的E.连胜的单个菌落干燥，种子液体LB培养LB琼脂上的大肠杆菌OP50细菌。你应该准备每60毫米的钢板至少有1毫升一夜之间OP50文化。 </li><li>广场OP50文化在37℃摇床，并允许细菌的生长过夜（文化应达到饱和）。 </li><li>佩莱OP50细菌纺纱在3500克为10分钟。 </li><li>取出上清和悬浮细菌的90％集中文化10X。 </li><li>吸取100μL，10倍浓缩OP50文化中心成为凝固NGM板。旋流板轻轻地摊开细菌培养，如果需要的话（理想的细菌应包括中部地区附近的板壁上的NGM）。尽量避免触摸枪头NGM的表面，在介质的表面缺陷允许进入NGM的蠕虫洞穴。 </li><li>保留过夜板在板凳上，让细菌培养，干燥的固体NGM（通常需要24小时左右）。 </li><li>干燥后，揭露板表面的紫外线剂量足以阻止细菌的生长。如果您使用的是一个Stratagene公司紫外线Stratalinker 2400： <ul><li>将板Stratalinker删除盖</li><li>关闭的大门，并开启了Stratalinker </li><li>按“能源” </li><li>输入&#39;9999&#39;使用键盘</li><li>按“开始” </li><li>约5分钟，板块将暴露在紫外线</li></ul></li><li>与紫外线杀死细菌板可存放在4 ° C的长达1个月倒挂。 </li></ol>第2部分：执行一个定时产蛋获得一个时代同步的动物人口在本节中，我们产生一个共同的孵化最新的蠕虫病毒的人口。这是通过允许繁殖活跃的成年人躺在一盘鸡蛋规定的时间内，让这些鸡蛋发展。 <ol><li> （可选）约20名年轻的成虫转移到新鲜无FUDR NGM板。离开板在25 ° C，以使蠕虫传播，并通过所有的细菌的食物吃。细菌的食物被消耗掉后，将进入新孵出的蠕虫被捕的dauer阶段的增长。你将开始看到周围的dauer幼虫在一个星期内，取决于你多少蠕虫转移到板最初的。可用于长达1个月的下一步骤的dauer幼虫。 </li><li> （可选）传输​​20至30的dauer幼虫没有FUDR新鲜NGM板。在食品的dauer幼虫存在将成为繁殖活跃的成年人，2天之内，并保持生殖活动及此后几天在25 ° C。 </li><li>将10至15繁殖活跃的成年人没有FUDR新鲜NGM板。这盘被称为定时产蛋（电话）板。 </li><li>留下电话板在25℃，6个小时，以便吨他蠕虫产卵。 </li><li>从电话钢板中取出成虫。板可目视检查之前消除成年人的鸡蛋。如果蠕虫没有奠定了足够数量的鸡蛋的电话板可以留下最多的前24小时删除成虫总在25 ° C。 </li><li>广场电话板在20 ° C，直到鸡蛋孵化的蠕虫已经开发的L4幼虫阶段（这通常需要2天，野生型线虫，但可以采取与发展缓慢的表型的菌株）。 </li></ol>第3部分：对分数的动物的寿命在本节中，我们按照从第2部分蠕虫人口的年龄同步，直到他们死去。蠕虫保持放大器/ FUDR板，以防止鸡蛋生产和细菌污染，被认为是死时，他们无法应对外部刺激。 <ol><li> L4幼虫转移到种子安培/ FUDR板。对于每一株或条件被测试，这是典型的设立与25-30蠕虫每盘2-3板。图片的C.线虫的生命阶段提供参考（图1）1 。 </li><li>广场安培/ FUDR板在20 ° C时24小时。 </li><li> 24小时后，在视觉上评估蠕虫，媒体和细菌。如果下列任何观察，蠕虫传输到新鲜安培/ FUDR板： <ul><li>蠕虫吃掉大部分细菌的食物。早在寿命实验蠕虫将有一个星期，以防止被耗尽的细菌转移1至2次。 </li><li>一个幼虫的大量存在。这通常是一个蠕虫被转移到为青壮年，而不是L4s安培/ FUDR板，能够打下一些鸡蛋，FUDR生效前的迹象。 FUDR将一般的幼虫防止日益充分的成年人，但偶尔有几个会成长为成年人，并成为与实验动物相混淆。 </li><li>直播/细菌滋生。一般来说，放大器和紫外线杀的组合将确保不活的细菌污染的实验，但偶尔会发生。 </li><li>真菌的生长是在媒体上观察到。如果早期发现足够的，真菌的生长通常可以切出使用枪头或锅铲。一旦它的增长要大于一个直径通常是更容易地转移到一个新的板块的蠕虫几毫米。 </li><li>使用的清扫范围，确定每一个蠕虫病毒是否是活着还是死了。 </li><li>轻轻敲击板。该蠕虫病毒是活的，如果它在攻丝移动。 </li><li>如果蠕虫不响应敲击头部区域板块，放大。 </li><li>轻轻拍打蠕虫的铂转移挑头。分数为死的蠕虫病毒通过移动它的头，如果它不响应。 </li><li>死亡的蠕虫可以从板块中删除。 </li></ul></li><li>记录日期和蠕虫的数量，还活着的和死的。 </li><li>返回板块至20 ° C。 </li><li>重复步骤3.3至3.6每2至3天，直到所有的蠕虫已经死亡。 </li></ol>第4部分：代表性的结果。 一种线虫的寿命实验中产生的原始数据是一个相应的数字还活着，并为每个测试应变死的蠕虫的日期列表。蠕虫每天死亡的人数通常是反转来计算的每一天活着的蠕虫的比例（图2A），作为一种生存曲线（图2B图形绘制;每天定时产蛋被认为是0天）。每个个人在研究蠕虫的寿命可以计算出从每天死亡的蠕虫计数，用来计算菌株间比较平均数和中位数的寿命。每一天活着的蠕虫数量的计数是不能直接使用在寿命分析。固体培养基上保持蠕虫偶尔会“逃离”，或爬上墙壁板或下方的媒体之一。每一天活着的蠕虫的数量可以被用来确定多少蠕虫在整个实验过程中出逃。蠕虫，逃离通常从分析中删除。为基准，典型的中位数为N2 的 C.寿命线虫野生型菌株对紫外线杀死细菌维持在20 ° C是从鸡蛋计算，约25天。 第5部分：解决方案<table border="0"><tbody><tr><td colspan="2"> 线虫的生长介质（NGM），100毫升： </td></tr><tr><td colspan="2">结合： </td></tr><tr><td> 0.3摹</td><td>氯化钠</td></tr><tr><td> 0.25摹</td><td>蛋白胨</td></tr><tr><td> 2摹</td><td>洋菜</td></tr><tr><td colspan="2"> 高压灭菌40分钟，放凉至55℃，然后添加： </td></tr><tr><td> 100μL， </td><td> 1个M硫酸镁4 </td></tr><tr><td> 100μL， </td><td> 5 mg / mL的胆固醇</td></tr><tr><td> 100μL， </td><td> 1中号氯化钙2 </td></tr><tr><td> 1.625毫升</td><td> 1.5米KPI pH值6.0 </td></tr><tr><td colspan="2">液体NGM的，可立即使用倒板或允许巩固和长期存储在室温</td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 卢里亚肉汤（LB），1L： </td></tr><tr><td> 10克</td><td> BactoTryptone </td></tr><tr><td> 5克</td><td>酵母提取物</td></tr><tr><td> 10克</td><td>氯化钠</td></tr><tr><td> 10毫升</td><td> 1米的Tris pH值8.0 </td></tr><tr><td> 1升</td><td>去离子水</td></tr><tr><td colspan="2">高压灭菌器，并在室温下储存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1个M 硫酸镁 300毫升： </td></tr><tr><td> 73.95摹</td><td> 硫酸镁 </td></tr><tr><td> 300毫升</td><td>去离子水</td></tr><tr><td colspan="2">高压灭菌器，并在室温下储存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 5 mg / mL的胆固醇，200毫升： </td></tr><tr><td> 1克</td><td>胆固醇</td></tr><tr><td> 200毫升</td><td> 100％的乙醇</td></tr><tr><td colspan="2">过滤消毒，并在室温下保存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1米氯化钙 ，500毫升： </td></tr><tr><td> 27.75摹</td><td> 氯化钙 </td></tr><tr><td> 500毫升</td><td>去离子水</td></tr><tr><td colspan="2">过滤消毒，并在室温下保存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1.5米KPI pH值6.0，1L： </td></tr><tr><td colspan="2">结合： </td></tr><tr><td> 31.4摹</td><td> KPI二元</td></tr><tr><td> 179.6摹</td><td> KPI二氢</td></tr><tr><td> 850毫升</td><td>去离子水</td></tr><tr><td colspan="2">加热边搅拌，让KPI溶解到溶液中。 10 N的氢氧化钠调节pH值至6.0。 </td></tr><tr><td colspan="2">加入去离子水至1 L。 </td></tr><tr><td colspan="2">高压灭菌器，并在室温下储存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1米三，pH值8.0： </td></tr><tr><td> 60.57摹</td><td>三</td></tr><tr><td> 400毫升</td><td>去离子水</td></tr><tr><td colspan="2">用HCl调节pH至8.0。 </td></tr><tr><td colspan="2">加入去离子水至500 ml。 </td></tr><tr><td colspan="2">过滤消毒，并在室温下保存。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 150毫米Fluorodeoxyuridine（FUDR），10毫升： </td></tr><tr><td> 0.3693克</td><td> FUDR </td></tr><tr><td> 10毫升</td><td>灭菌去离子水</td></tr><tr><td colspan="2">储存在-20 ° C。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 100毫克/ mL氨苄青霉素（AMP），10毫升： </td></tr><tr><td> 1克</td><td>氨苄青霉素</td></tr><tr><td> 10毫升</td><td>灭菌去离子水</td></tr><tr><td colspan="2">储存在-20 ° C。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 50毫克/毫升羧（CARB），10毫升： </td></tr><tr><td> 500毫克</td><td>羧</td></tr><tr><td> 10毫升</td><td>灭菌去离子水</td></tr><tr><td colspan="2">储存在-20 ° C。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2"> 1米异丙基β- D -硫代半乳糖苷（IPTG），10毫升： </td></tr><tr><td> 2.38摹</td><td>经IPTG </td></tr><tr><td> 10毫升</td><td>灭菌去离子水</td></tr><tr><td colspan="2">储存在-20 ° C。 </td></tr></tbody></table><img src="/files/ftp_upload/1152/Figure%201.png" alt="图1" /> 图1 C.明场图像。 线虫的生命阶段，包括卵，幼虫的四个阶段（L1 - L4），和成人。所有面板显示除右下角，这显示了一个成年男性（木（1988年）的图像）雌雄同体。 <img src="/files/ftp_upload/1152/Figure%202.png" alt="图2" /> 从A C.代表性的结果图2。 线虫的寿命实验比较应变包含在DAF - 2基因突变的野生型菌株N2 。 （A）表显示收集的数据，包括天数，因为蠕虫是鸡蛋，每天观察死亡蠕虫数，和原始样品的剩余百分比活着的每一天（从死虫的日常计数计算） 。 （二）生存曲线对应的寿命（一）提供的数据。 <imgSRC =“/ files/ftp_upload/1152/Figure 3.JPG”ALT =“图3”/&gt; 脂褐素的代表比较年轻人与老年人之间的 成年人C.图 3。 线虫 。明场图像显示在左侧通道的DAPI荧光图像的权利。前面板显示，4天的老蠕虫和底部面板显示了11天的老蠕虫（从鸡蛋衡量）。

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+luminescence+spectroscopy+of+fluorescence+changes+during+aging+in+Caenorhabditis+elegans.">Total luminescence spectroscopy of fluorescence changes during aging in Caenorhabditis elegans.</a> Biochemistry. 21, 4089-4095 (1982).</li><li>Klass, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging+in+the+nematode+Caenorhabditis+elegans:+major+biological+and+environmental+factors+influencing+life+span.">Aging in the nematode Caenorhabditis elegans: major biological and environmental factors influencing life span.</a> Mech Ageing Dev. 6, 413-429 (1977).</li><li>Steinkraus, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+restriction+suppresses+proteotoxicity+and+enhances+longevity+by+an+hsf-1-dependent+mechanism+in+Caenorhabditis+elegans.">Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans.</a> Aging Cell. 7, 394-404 (2008).</li></ol>

长寿的遗传控制已被广泛研究， 在 C 线虫 ，主要是由于与寿命可确定的易用性和快速性。在这篇文章中讨论的协议描述为获得重现生命跨度的数据， 在 C的基本框架也可以应用于线虫和relatednematode物种。做一些简单的改动，这些程序可适应各种条件下的寿命来衡量。在这里，我们将讨论几种常见的变化，包括活的细菌，细菌剥夺限制饮食，RNA干扰（RNAi），和无毒品NGM?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0145-17-0 (214530)</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>Reagent</td>
<td>MidSci</td>
<td>0339</td>
<td>&nbsp;</td>
<tr>
<td>BactoTryptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0123-17-3 (211705)</td>
<td>&nbsp;</td>
<tr>
<td>BactoPeptone</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0118-17-0 (211677)</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9699248 (1332-01)</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>C0109</td>
<td>&nbsp;</td>
<tr>
<td>Cholesterol</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>C75209</td>
<td>&nbsp;</td>
<tr>
<td>Fluorodeoxyuridine (FUDR)</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0503</td>
<td>&nbsp;</td>
<tr>
<td>Isopropyl &#x03B2;-d-Thiogalactopyranoside (IPTG)</td>
<td>Reagent</td>
<td>Gold BioTechnology (GBT)</td>
<td>I2481C5</td>
<td>&nbsp;</td>
<tr>
<td>KPi Dibasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087862 (3252-01)</td>
<td>&nbsp;</td>
<tr>
<td>KPi Monobasic</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>5087861 (3246-01)</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>Reagent</td>
<td>Fisher Scientific (JT Baker)</td>
<td>NC9561800 (2500-01)</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>Reagent</td>
<td>Fisher Scientific</td>
<td>S251</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>Reagent</td>
<td>Fisher Scientific (BD Diagnostic Systems)</td>
<td>DF0886-17-0 (288620)</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

measuring caenorhabditis elegans life span on solid media

老化是一种退化性的过程，特点是逐渐恶化的细胞成分和细胞器造成的死亡率。线虫<em&gt;线虫</em用来研究老化生物学的一个主要模式出现。因为随着年龄的增加，几乎每一个生物子系统进行功能下降，寿命是利益的考虑老化率时的主要终点。线虫的寿命通常定义为一种动物的天数仍然对外界刺激的的反应。线虫可以在液体介质中或板固体培养基上，并测量两个条件下的寿命的开发技术已。这里我们提出一个广义的协议和测量寿命固体线虫的生长介质保持线虫喂食的紫外线杀死细菌的饮食。这些程序可以很容易地适应各种常见的条件下测定的寿命，包括饮食活的细菌，限制饮食，和RNA干扰组成。

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Life Span on Solid Media at JoVE.com

Measuring Caenorhabditis elegans Life Span on Solid Media

在这篇文章中，我们提出了一个测量寿命固体培养基上保持线虫用紫外线灭活的细菌性食物的一般协议。

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 ...

measuring-caenorhabditis-elegans-life-span-on-solid-media

Research

JoVE Journal

Biology

Measuring Caenorhabditis elegans Life Span on Solid Media

29.7K 次观看.  University of Washington. 在这篇文章中，我们提出了一个测量寿命固体培养基上保持线虫用紫外线灭活的细菌性食物的一般协议。

视频: Measuring Caenorhabditis elegans Life Span on Solid Media

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 ...

科研

生物学

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 ...

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

实心板为基础的饮食限制在线虫</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

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 ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

培养<em&gt;秀丽隐杆线虫</em &gt;在无菌液体介质与创造转基因蠕虫通过微粒轰击

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

使用刺激拉曼散射显微镜对 卡诺哈布迪炎电子人 中的脂质存储动力学进行无标签成像

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...