我们要感谢S.塔内加- Bageshwar，K. Beifuss，S. Kedroske，和HC。萧为有益的讨论。这项工作是由从分子和细胞医学TAMHSC系的启动资金。该化合物的范围和旋转盘购部门和TAMHSC院长办公室提供的资金。一些菌株提供了线虫遗传学中心，这是由国家研究资源中心资助。 pRF4（ROL - 6（su1006））答:消防的礼物。

1。 DII制备染色<ol><li>准备的20毫克/毫升DII（Biotium公司，公司，海沃德，CA）在DMF原液。 DII是光敏感，所以保护光箔包装DII。 </li><li>每个人口0.6μLDII股票加入到399.4微升M9的创建工作的DII稀释。这应该给最后的30微克/毫升DII在M9的工作液。这可以扩展多个种群同时染色。盾DII在铝箔包装管（S）。 </li></ol> 2。线虫的制备<ol><li>包含一个未被污染的线虫人口使用60毫米的钢板。洗轻轻摇动整个板面的液体的圆周运动，放松所有的幼虫和成年动物，动物板，解决了0.5％的Triton X - 100的使用M9的缓冲区。转移到无菌1.5毫升管洗。 </li><li>立即降速在2000转每分钟30秒的动物。取下并丢弃很多SUPernatant尽可能在试管底部，而不会干扰动物的质量。 </li><li>为了减少残留的TRITON X - 100，冲洗动物使用M9的缓冲区，旋转，去除上清液。重复此步骤一次。 </li><li>管和涡简要加入400μL的工作DII M9的解决方案，以重悬在溶液中的动物。 </li><li>摇管在20 ° C水平为3个小时在350 RPM轻保护环境。如果需要的话，动物可以被孵育染色16小时。 </li><li>为了减少未结合的染料量，自旋向下的动物，在2000转，持续20秒。删除尽可能上清液丢弃，而不会干扰动物的质量。 </li><li>在400μLM9的缓冲区，并倒到无细菌的接种与OP50 E.一个NGM琼脂板部分液体悬浮动物大肠杆菌 。让动物恢复至少30分钟。在恢复时间的动物应该爬远离DII染色液体到食品。这一步免费直接投资收益减少背景荧光。 </li></ol> 3。安装和观察标本<ol><li>使用高压灭菌器或微波熔体4％水琼脂。 </li><li>创建可重复使用的垫片，可用于确保琼脂板厚度均匀，分层两件实验室磁带载玻片上。共两个间隔的​​幻灯片。 </li><li>两个间隔幻灯片之间安排一个洁净的载玻片。移液器熔化的4％到洁净的载玻片中心琼脂约150μL（四滴）。迅速覆盖使用一个额外的幻灯片的形式琼脂垫熔化的琼脂。小心地取下封面幻灯片，安装幻灯片的顶部为中心的垫。 </li><li>移液器约5μL线虫麻醉（100微米 - 1毫米左旋咪唑，例如，）上垫。 </li><li>山8 - 12只动物在麻醉剂，并与显微镜的盖玻片覆盖。 </li><li>使用装有至少40倍objecti的化合物或共聚焦显微镜观察动物<html> VE和一个DsRed的/ TRITC（或其他兼容）过滤器。激发最大的投资收益为549纳米，其最大发射约束染料（Biotium公司，公司，海沃德，CA）是565纳米。 </li></ol> 4。代表性的成果 DII污渍角质层的野生型和突变株C.线虫 。表皮表面含有环空中圆周沟分开，并在某些阶段，纵脊alae。每个发展阶段有2个不同成分的表皮结构。 alae和环空中的山脊或沟荧光染色，取决于表面成分，整个幼虫和成年阶段，仍然可见使用这种方法恢复后的一天。有时观察到的背景荧光斑点（图2F，G）的，但不是常规（图1，图2A - E，H，我）。所有图像旋转盘共聚焦或时指出，广角（WF）复合显微镜。 耳鼻喉科"&gt; <img alt="图1" src="/files/ftp_upload/3362/3362fig1.jpg" /> 图1。 DII污渍角质层和环境接触的神经元。 L2阶段的动物。 630x放大倍率。马赛克图像部分使用iVision - Mac软件（BioVision的技术，位于宾夕法尼亚州Exton）被抓获。使用Adobe Photoshop CS3（Adobe系统公司，加利福尼亚州圣何塞）加入图像。比例尺= 10微米。 DII还荧光污渍amphid phasmid感觉神经元的头部和尾部分别为（箭头标记一些）。 <img alt="图2" src="/files/ftp_upload/3362/3362fig2.jpg" /> 图2。 DII C.荧光污渍线虫角质层后胚胎发育的各个阶段 。 630x放大倍率。比例尺= 10微米。野生型动物染色）L1，B）二级C）的dauer，D）三级，E）L4;和F）成人阶段。在L1和的dauer动物alae和环形脊荧光染色（A，C）。 DII污渍二级动物的环形脊（二）。环形沟L3和L4动物（D，E）染色。在alae沟和环空中成年动物（FH）的染色。 Alae是由二，五，或三个脊（L1，的dauer，或成年动物，分别）运行长度的动物（箭头）16。环空中创建圆周周围的动物脊（箭头，F和J）。角质层成人突变动物显示温和的表皮组织缺损（GH）。 G）alae的脊不连续（WF）。高）编外alae脊融合和分支或分叉（WF）。胶原蛋白基因的突变体表现alae和环形组织缺损（IJ）。 i）在过表达转基因动物pRF4（ROL - 6（su1006）），alae谎言脊动物的长度角度。 J）在转基因动物的过度表达pRF4（ROL - 6（su1006））环空中显示一个不规则的图案。 <img alt="图3" src="/files/ftp_upload/3362/3362fig3.jpg" /> 图3。为extern人的形态结构，照亮了DII染色。630x放大倍率。比例尺= 10微米。 DII还突出等外观特征，包括一）成人雌雄同体外阴，B）的成年雄性的尾巴射线和风扇和C）雌雄同体的尾穗（在这种突变的背景分叉）。 <img alt="图4" src="/files/ftp_upload/3362/3362fig4.jpg" /> 图4。角质层需要更长的时间比环境接触的神经元与DII染色。 630x放大倍率。比例尺= 10微米。经过两个小时的染色，amphid（未显示）和phasmid感觉神经元（箭头）有足够的染色。相比之下，年轻动物的角质层只是部分补丁（箭头）染色。 <table border="1"><tbody><tr><td> 洗 </td><td> 染色解决方案（DII） </td><td> 孵育时间 </td><td> 角质层染色 </td></tr><tr><td> M9的± 0.5％TRiton X - 100 </td><td> M9 + 0.5％的Triton X - 100 </td><td> 2小时</td><td>没有</td></tr><tr><td> M9 + 0.5％的Triton X - 100 </td><td> M9 + 0.5％的Triton X - 100 </td><td> 3小时</td><td>没有</td></tr><tr><td> M9 + 0.5％的Triton X - 100 </td><td> M9 </td><td> 2小时</td><td>部分</td></tr><tr><td> M9 + 0.5％的Triton X - 100 </td><td> M9 </td><td> 3小时</td><td>是</td></tr><tr><td> M9 + 0.5％的Triton X - 100 </td><td> H 2 O </td><td> 2小时</td><td>部分</td></tr><tr><td> M9 + 0.5％的Triton X - 100 </td><td> H 2 O </td><td> 3小时</td><td>是</td></tr></tbody></table> 表1。表皮染色，在不同的条件下，各种孵化的解决方案和时间进行了测试，以优化在动物表皮染色。 H 2 O，无菌蒸馏水。部分染色表明幼虫角质层片状染色（图4），虽然成人角质层污渍的彗星onsistently。

<ol>
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我们什么都没有透露。

这里介绍DII的染色方法，允许一个相对快速和方便的方法来可视化的角质层线虫。再利用和优化常用的图像环境暴露15,17感觉神经元的方法，直接投资收益可用于荧光染色alae和环状结构（图1和2），以及外阴，男性的尾巴，和雌雄同体的尾穗（图3）。我们发现，洗液和时间的影响的直接投资收益的能力，始终染色的角质层（表1，图4）。 DII染色环境暴露的神经元的方法使用两个小时的孵育时间M9 TRITON X - 100 15。 M9与表面活性剂的初始洗TRITON X - 100有助于消除从表皮表面的污染和防止结块动物。然而，在同一个缓冲区染色液孵化三个小时的动物防止染料染色的角质层（表1）。这可能是引起血脂线虫表面上被剥夺了在洗涤剂溶液治疗。潜伏期的动物DII污渍角质层的水，表明M9盐溶液不是DII角质层染色的要求。虽然水为基础的协议是神经元DII染色15的建议，我们发现，动物治疗这种方式往往出现不健康的。洗衣机在0.5％的动物简要Triton X - 100在M9的的，在染色液三个小时的孵化与M9的表皮结构，提供一致的染色和维护动物福祉。 动物可以获救后的观察和维护，允许直接下游分析。这种方法是一种方便的工具，它可以在许多研究中使用，包括但不限于，表皮的分泌和组织，表皮细胞的发育，heterochronic基因的途径，和线虫的演变。

<table border="1"><tbody><tr><td> 试剂 </td><td> 同义词 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DII </td><td> 1,1&#39; - 双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐</td><td> Biotium公司，公司</td><td> 60010 </td><td>股票稀释: 20 mg / ml的二甲基甲酰胺 工作液:30毫克/毫升</td></tr><tr><td>二甲基甲酰胺</td><td>二甲基甲酰胺</td><td> Sigma - Aldrich公司，公司</td><td> D4551 </td><td></td></tr><tr><td>海卫一 X - 100 </td><td> Octylphenoxypolyethoxyethanol </td><td>厂商VWR International，LLC。 </td><td> EM - 9410 </td><td></td></tr><tr><td> M9 </td><td> 22MM KH 2 PO 4，4200万娜2 HPO 4，86mm的氯化钠，硫酸镁1MM 4 </td><td></td><td></td><td></td></tr><tr><td> NGM &lt;/ TD&gt; <td>线虫的生长介质</td><td> IPM的科学，公司</td><td> 11006-501 </td><td>可以准备以下NGM琼脂协议18 </td></tr><tr><td>琼脂</td><td></td><td>电解二氧化锰化工公司</td><td> 1.01614 </td><td> 4％水</td></tr><tr><td>左旋咪唑</td><td>盐酸左旋咪唑</td><td> Sigma - Aldrich公司，公司</td><td> 31742 </td><td> 100微米 - 1毫米所需的左旋咪唑</td></tr><tr><td>显微镜载玻片</td><td></td><td>厂商VWR International，LLC。 </td><td> 16005-106 </td><td></td></tr><tr><td>显微镜盖玻片</td><td></td><td>厂商VWR International，LLC。 </td><td> 16004-302 </td><td></td></tr><tr><td>复方范围</td><td></td><td>卡尔蔡司公司</td><td> A1m </td><td>利用的目标，以满足实验的需要</td></tr><tr><td> TRITC或其他兼容滤波器</td><td> ＆NBSP; </td><td>色度科技股份有限公司</td><td> 49005 </td><td> （ET - DsRed的TRITC/Cy3）溅射过滤器设置</td></tr></tbody></table>

visualization of caenorhabditis elegans cuticular structures using the lipophilic vital dye dii

角质层的C.线虫是一种高度耐药的结构，周围的动物 1-4外部。角质层不仅可以保护环境的动物，但也决定体形和蠕动4-6次的作用。由表皮细胞分泌的几层组成的角质层，包括最外层的脂质层7。 在角质层的圆周脊称为环空中的模式动物的长度， 目前在8发展的所有阶段。 Alae有纵脊目前是在特定的发展阶段，包括L1的dauer，和成人阶段2,9。影响表皮胶原组织的基因突变可以改变表皮结构和动物的身体形态 5,6,10,11 。虽然使用DIC的光学显微镜复合表皮成像是可行的，目前的方法，突出表皮结构包括荧光12％的转基因表达，抗体染色，电子显微镜1。也被用来标记麦胚凝集素（WGA）的可视化表皮糖蛋白，但受限于解决更精细的表皮结构 14 。使用荧光染料染色表皮表面已被观察到，但从来没有在细节 15的特点。我们提出了一个方法来可视化角质层在现场 C. 线虫使用红色荧光亲脂性染料DII（1,1&#39; -双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐），也就是通常用于在 C 线虫可视化环境接触的神经元。这是一个简单可靠的方法，高分辨率的荧光可视化的环空中，alae，外阴，男性的尾巴， 在 C和雌雄同体的尾穗DII染色的优化协议线虫 。

Watch this Scientific Journal Video about Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI at JoVE.com

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

我们提出了一个可视化生活角质层的方法<em&gt; C。线虫</em&gt;使用红色荧光亲脂性染料DII（1,1&#39; - 双十八烷基- 3，3,3&#39;，3&#39; - tetramethylindocarbocyanine高氯酸盐），这是常用的<em&gt; C。线虫</em&gt;可视化环境接触的神经元。这种优化的协议，alae和环形表皮结构是由DII染色和使用复合显微镜观察。

可视化线虫角质层结构的亲脂性的重要染料，直接投资收益

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

visualization-caenorhabditis-elegans-cuticular-structures-using

Nanyang Technological University

Research

JoVE Journal

Biology

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

17.0K 次观看.  Texas A&M University System Health Science Center, College of Medicine. 我们提出了一个可视化生活角质层的方法 C。线虫使用红色荧光亲脂性染料DII（1,1' - 双十八烷基- 3，3,3'，3' - tetramethylindocarbocyanine高氯酸盐），这是常用的 C。线虫可视化环境接触的神经元。这种优化的协议，alae和环形表皮结构是由DII染色和使用复合显微镜观察。

视频: Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

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 ...

科研

生物学

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 ...

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

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

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

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

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

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 ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

DiI灌注作为血管可视化的方法<em&gt;墨西哥恶性肿瘤</em

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

卡诺尔哈布炎细胞外囊泡的纯化与分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

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, ...

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 ...

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and sequencing. The traditional proteinase K-based methods for genomic DNA extraction from C. elegans take several hours. Commercial extraction kits that effectively break open the C. elegans cuticle and extract genomic DNA are limited. An easy, faster (~15 min), and cost-efficient method of extracting C. elegans genomic DNA that works well for classroom and research applications is reported here. This DNA extraction method is optimized to use single or a few late-larval (L4) or adult nematodes as starting material for obtaining a reliable template to perform PCR. The results indicate that the DNA quality is suitable for amplifying gene targets of different sizes by PCR, permitting genotyping of single or a few animals even at dilutions to one-fiftieth of the genomic DNA from a single adult per reaction. The reported protocols can be reliably used to quickly produce DNA template from a single or a small sample of C. elegans for PCR-based applications.

Small-Scale Extraction of Caenorhabditis elegans Genomic DNA

Presented here is a description of the straightforward and relatively rapid isolation of Caenorhabditis elegans genomic DNA from one or a few animals using a commercially available tissue kit. The resulting gDNA preparation is a suitable template for PCR.

小规模提取 秀丽隐杆线虫 基因组DNA

Genomic DNA extraction from single or a few Caenorhabditis elegans has many downstream applications, including PCR for genotyping lines, cloning, and ...