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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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?...

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 border="0" cellpadding="0" cellspacing="0">
	<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 60x15mm</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10ml</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 colspan="2">Discovery v8 stereo zeiss microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well clear microtiter plate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>flcn-1 RNAi source</td>
			<td colspan="2">Ahringer Library</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...

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

Research

JoVE Journal

Biology

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

15.3K 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

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

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

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 ...

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, ...

Analysis of Oxidative Stress in Zebrafish Embryos

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

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

在斑马鱼胚胎氧化应激分析

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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

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 ...