作者想承认，AGAUR（博士奖学金到劳拉Fontrodona），MICINN（PTA项目配套蒙特塞拉特肝门德拉·里瓦），研究所的Salud的卡洛斯三世（米格尔Servet计划支持朱利安Cerón），玛丽·居里表意文字，ISCIII和IDIBELL融资实验室。

1。议定书:11漂白培养蠕虫 C.人口众多线虫可以得到培养他们无论是在液体介质或固体介质中板。它们通常生长在固体NGM的（线虫的生长介质）和美联储与E.大肠杆菌的细菌，它被添加到的板块无论是活着还是死了（由紫外线12死亡，13热或冷14）。最常见的程序使用现场OP50 E。大肠杆菌 ，这是在尿嘧啶合成缺陷，并不能长满成厚厚的一层掩盖蠕虫。 <ol><li>混合3克盐，17克和琼脂，蛋白胨2.5Ğ加975毫升H 2 O高压灭菌50分钟。 </li><li>烧瓶冷却至55°C </li><li>新增的1 M 氯化钙 ，1毫升1毫升5毫克/毫升乙醇胆固醇，1毫升的1 M 硫酸镁 25毫升的1 M KPO 4缓冲（所有的人，但胆固醇以前autoclavED）。 </li><li>使用无菌程序免除成Petri板NGM的解决方案;板填补了其体积的2/3。 </li><li>一旦干燥，最好是离开板在室温下2-3天前使用污染物检测。准备OP50 五连胜从甘 ​​油股票大肠杆菌 （OP50可以从线虫遗传学中心获得）。 </li><li>挑单菌落在LB隔夜增长在37°C，搅拌。 </li><li>让多余的水分蒸发板层流中删除盖子，并用无菌巴斯德吸管OP50板块的中心。 </li><li>允许OP50 E。大肠杆菌草坪增长8小时，在室温或在37°C过夜。 </li><li>加入所需数量的蠕虫板（如果在37°C孵育应在使用前在室温下冷却板）。 </li></ol>提示: <ul><li>浇注相同数量的媒体确保在用吸管或泵分配器板相同体积的琼脂板，并有利于板块的转移而不需要重新调整的体视显微镜。 </li><li>几个星期后，准备在容器中存放在室温或4°C时，可用于板（去籽，与细菌的非种子选手） </li><li>避免电镀板边缘的细菌。如果草坪延伸到的板块边缘的蠕虫可能会爬上两侧，干死。 </li><li>蠕虫活得更长，如果种子的盘子上的细菌是已经死了15。 </li></ul> 2。协议B:治疗用碱性次氯酸钠溶液（"漂白"）11 漂白技术用于同步C。线虫幼虫在第一阶段的文化（第一级）。该方法的原理在于蛋壳保护E的事实蠕虫敏感漂白IT mbryos从。用碱性次氯酸钠溶液处理后，胚胎培养在液体培养基中没有食物，使孵化，但防止进一步发展。 <ol><li>允许蠕虫生长，直到成年阶段。 </li><li>洗涤板与M9的缓冲区，恢复在15毫升管妊娠成人。 </li><li>离心2分钟在400xg在室温（〜1500标准表离心机的转速）和弃上清丸蠕虫。 </li><li>执行1-3洗涤，直到缓冲区出现明确的细菌。 </li><li>添加所需的漂白液（ 见表一 ），并鼓动一些分钟（成人组织的破坏，应在解剖显微镜下监测和反应停止时，成人的痕迹仍清晰可见，这通常需要3至9分钟，取决于几个问题，如蠕虫病毒颗粒的体积在讨论中提到，）（ 图3）。 </li><li>停止反应，加入M9的buffer来填补管。 </li><li>快速离心1分钟（因为治疗可能仍处于活动状态），在400 XG，弃上清。 </li><li> M9的缓冲区填充管清洗颗粒三次。 </li><li>加入1毫升的M9缓冲沉淀，或以非种子选手NGM的板放置鸡蛋，孵化所需的温度轻轻摇动。应提供适当的通风（ 图4）。 </li></ol>提示: <ul><li>有不同的漂白解决方案，选择一个工作在你的手中（ 表1，图1）。 </li><li>可以回收报废一块的X光片，如软质材料的琼脂表面板已经产卵。 </li><li>成年动物太多的遗体，可能会损害同步，因为它们构成了一个刚孵化的幼虫的食物供应。 </li><li>较高的温度略有加快发展，这是不方便，我f任何蠕虫跳过同步在较高的温度，因为在发展中的同步和不同步的蠕虫之间的差异将更大。 </li><li>漂白液必须在使用前进行。此外，漂白剂失去效力后，一直开放的同时，部分原因是由于其光敏性。我们建议每个新瓶分装成小的琥珀色瓶，以防止这种损失和尽量减少暴露在光线下。 </li></ul> 3。协议的C:蠕虫电镀<ol><li>等待12至24小时内（完成胚胎发育的时间取决于温度）进行漂白后恢复离心400 XG（2分钟）的蠕虫病毒。 </li><li>弃上清，所需的板种蠕虫，让剩余的液体干。 </li><li>在所需的温度下放置板。 </li></ol>提示: <ul><li> L1 M9的缓冲区幼虫可保持在15°C间摇摆至少一个星期的Wi大地明显改变。 </li><li>要小心计算时将种子的蠕虫可能会耗尽，因为太多的食物比预期更快地，毁了你的实验。约500 L1可以达到55毫米板成年食品不运行。 </li></ul> 4。协议ð:C。线虫观察 D.1中Nomarski观察微分干涉对比显微镜是光学显微镜的照明技术，用于提高在未染色透明标本的对比。 Nomarski这个词是指棱镜后，他的发明者的名字命名的。通过观察动物活着，我们能够从固定的唯一改变动物的生理研究。此外，因为没有添加固定液，荧光标记物，可在体内观察。这一事实，并融合到一个感兴趣的基因的荧光标记物的可能性是可行的，按照进程在W更富研究蛋白质可能参与。通过此协议中所描述的技术，可以观察到，不仅活虫，但它们也可以被再次回收，镀。 琼脂垫准备（使用前）: <ol><li>准备2％琼脂糖水和融化。保持融化在65°C。 </li><li>将两个幻灯片，与他们一块在第三，清洁的幻灯片双方的磁带。 </li><li>使用巴斯德吸管的地方，干净的表面上的琼脂下降。 </li><li>琼脂覆盖与垂直的三个幻灯片上放置另一块干净的幻灯片。 </li><li>轻轻按下，使琼脂下降磁带垫片的厚度被夷为平地。 </li><li>琼脂凝固后，轻轻地拉出录音幻灯片和一个相对滑动的其他分开的两个其余幻灯片。 </li></ol>安装活体动物<ol start="7"><li>地方1滴（10微升），左旋咪唑1MM或钠叠10-30毫米到ţ他的垫中心。 </li><li>转移到动物使用蠕虫采撷下降的。 </li><li>轻轻地放置在动物盖玻片和修复双方在一些指甲油或硅。 </li></ol>提示: <ul><li> 2％琼脂糖保持在4°C等分</li><li>融化的琼脂糖凝胶可保持在65°C，至少一天。 </li><li>注:左旋咪唑是一种尼古丁受体激动剂引起痉挛肌麻痹16。 </li><li> 谨慎，叠氮化钠是剧毒！ </li></ul> D.2乙醇固定和DAPI染色这里描述的协议表示用DAPI染色蠕虫的快速路，但是因为一些蠕虫dissecation结构可能会出现一些改变。还有其他一些方法来解决以前的蠕虫，如卡诺，更好地保存完整的蠕虫17的溶液或甲醛固定DAPI染色。 乙醇固定（18日修改） <ol><li>地方〜10μLM9的缓冲区，在显微镜幻灯片（或水）。 </li><li>使用蠕虫挑仔细将下降10-25蠕虫。 </li><li>如果不使用滤纸或微吸管去除尽可能多M9尽可能缓冲，消除蠕虫，或让他们干。 </li><li>加入10μL〜90％的乙醇，并让它干。 </li><li>重复步骤4一次或两次。 </li></ol> 4&#39;,6-diamidino-2-苯基吲哚（DAPI）的染色<ol start="6"><li>所需的安装媒体的终浓度2 ng /μl的混合的DAPI。 </li><li>一旦乙醇已完全蒸发，增加7μL的DAPI:混合安装媒体。 </li><li>放置盖玻片和修复双方在一些指甲油或硅。幻灯片将是观察约5分钟后的DAPI除了准备。 </li></ol>提示: <ul><li>安装MEDIA包含甘油，所以少量就足以覆盖整个筹备。 </li><li>存在着各种各样的商业安装媒体（Fluoromount或延长，例如），其质量和价格取决于多久你想储存您的样本。 </li><li> 要小心的DAPI是一个已知的诱变剂，它结合丰富的地区，在DNA强烈。 </li></ul>食谱 线虫的生长介质（NGM的） 琼脂1.7％（W / V） 50 mM氯化钠 0.25％（W / V）蛋白胨 1毫米氯化钙2 5微克/毫升胆固醇 25毫米KPO 4 1毫米硫酸镁4 M9的缓冲区 22MM锦2 PO 4计 42毫米的Na 2 HPO 4 86毫米氯化钠 1毫米硫酸镁4 漂白的解决方案测试 <table border="1"><tbodY&gt; <tr><td></td><td> 配方＃1 </td><td> 配方＃2 </td><td> 配方＃3 </td><td> 配方＃4 </td><td> 配方＃5 </td></tr><tr><td>水（毫升） </td><td> 2.75 </td><td> 3.5 </td><td> 0.5 </td><td> 0.5 </td><td> 1.5 </td></tr><tr><td>氢氧化钠（毫升） </td><td> 1.25（1M） </td><td> 0.5（5M） </td><td> 2.5（1M） </td><td> 2.5（2M） </td><td> 2.5（1M） </td></tr><tr><td>次氯酸钠300 4％（毫升） </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 2 </td><td> 1 </td></tr><tr><td>总计（毫升） </td><td> 5 </td><td> 5 </td><td> 4 </td><td> 5 </td><td> 5 </td></tr></tbody></table>本文的表一，不同的漂白液配方进行测试。食谱＃3和＃4的2倍，并应增加M9相同体积。 ＃1，＃2和＃5的食谱此前已报道2，11，19。最终浓度:0.25M 1氢氧化钠，次氯酸钠~~ 0.8％<html> ＃2的NaOH，0.5M，次氯酸钠〜0.8％，0.625M氢氧化钠，次氯酸钠〜1％氢氧化钠1，＃4男，次氯酸钠〜1.6％，＃5氢氧化钠0.5M，次氯酸钠〜0.8％。 5。代表结果<img src="/files/ftp_upload/4019/4019fig1.jpg" alt="图1" /> 图1。比较五种不同的漂白解决方案，在两个不同的孵育时间。与M9的两次冲氮气蠕虫被分成5 15包含每个漂白液的毫升锥形管。管大力动摇和1毫升转移到新管与M9在指定的时间后停止反应。经过漂白程序，蠕虫病毒在20°C孵育24小时，用1毫升的M9。在每一种情况下，较低的图片刚刚经过漂白，上图，24小时后采取的。 _upload/4019/4019fig2.jpg"ALT ="图2"/&gt; 图2。漂白液的温度会影响治疗效果。相同体积的N2蠕虫与先前冷藏冰20分钟，同时保持在25°C的解决方案相同的漂白漂白。两列左侧显示图片刚过漂白。经过治疗蠕虫在20°C孵育15毫升锥形管24小时，1毫升的M9。 24小时后列右侧显示​​图片。 <img src="/files/ftp_upload/4019/4019fig3.jpg" alt="图3" /> 图3。比蠕虫病毒颗粒:碱性次氯酸钠培养时间，影响治疗效果，蠕虫颗粒50，100，250和500μL孵育2毫升漂白3，第6和第9分钟的解决方案＃3。 2，孵化后在20°C孵化的L1，死胚胎和成人碎片遗骸进行量化<html> M9的缓冲区与1毫升15毫升锥形管4个小时。约三融合55毫米，成虫板需要获得100μl的蠕虫病毒颗粒。 <img src="/files/ftp_upload/4019/4019fig4.jpg" alt="图4" /> 图4。 C 的孵化和生存所需的适当的曝气线虫的胚胎。100μl的颗粒N2的蠕虫漂白6分钟，1，5年或10毫升，15毫升锥形管培养指定M9的24小时，在20°C。图的上半部分显示文化的图片，24小时后，其中箭头表示并没有孵化的蛋。在底部，有一个图，描绘幼虫（浅灰色）和死胚（深灰色）48小时后，在规定条件漂白。 tp_upload/4019/4019fig5.jpg"ALT ="图5"/&gt; 图5。 C 的生命周期线虫 。一个 。蠕虫在不同阶段的大致时间。小时要求达到根据每个阶段的温度（20修正）。Nomarski（上）和DAPI（下）在指定的发育阶段，不同的蠕虫图片。在每个阶段的最显着的特点是放大的L1:箭头指示体性腺及生殖系的前体早期L4:黑色箭头（Nomarski）表示开发外阴白色箭头表示（DAPI）的两个性腺武器的半山 。 晚L4:箭头表示在所谓的圣诞树阶段的发展外阴青年:黑色箭头表示子宫内的胚胎，箭头点到精囊，白色箭头指示卵妊娠成人:箭头（DAPI）的指出受精胚胎。精囊的DAPI图像中的箭头表示。 <img src="/files/ftp_upload/4019/4019fig6.jpg" alt="图6" /> 图6。外阴形态，在L3，L4和成人阶段。在L3阶段，只有一小管腔形成外阴可以观察到。在L4，这管腔扩大，形成所谓的"圣诞树"。在成人外阴已经关闭。黄线表示在这三个阶段的外阴位置。 <img src="/files/ftp_upload/4019/4019fig7.jpg" alt="图7" /> 图7。 DAPI染色，在L3，L4年初，在L3 L4和成人阶段。，胚芽线被拉长。在L4，性腺武器目前U形形态。在后期的L4阶段精子可以被观察到在性腺远端部分。青壮年提出的卵母细胞。成人生殖系卵母细胞和胚胎。黄线在日delimitate胚芽线é不同规定的阶段。 <img src="/files/ftp_upload/4019/4019fig8.jpg" alt="图8" /> 图8。C。线虫在15和25°C氮气蠕虫的发展进行了漂白，隔夜培养在M9和搅拌在15°C间，转移到板和指定的时间，在规定的温度下生长。

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U.S.A. 105, 19839-19344 (2008).</li><li>Voisine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+therapeutic+drugs+for+huntington's+disease+using+Caenorhabditis+elegans.">Identification of potential therapeutic drugs for huntington's disease using Caenorhabditis elegans.</a> PLoS One. 2, e504 (2007).</li><li>Garigan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tissue+aging+in+Caenorhabditis+elegans:+a+role+for+heat-shock+factor+and+bacterial+proliferation.">Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation.</a> Genetics. 161, 1101-1112 (2002).</li><li>Aceves, J., Erlij, D., Martinez-Maranon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+the+paralysing+action+of+tetramisole+on+Ascaris+somatic+muscle.">The mechanism of the paralysing action of tetramisole on Ascaris somatic muscle.</a> Br. J. Pharmacol. 38, 602-607 (1970).</li><li>Shaham, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+cell+biology.">Methods in cell biology.</a> Wormbook. , (2006).</li><li>Pepper, A. S., Killian, D. J., Hubbardm, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+Caenorhabditis+elegans+glp-1+mutants+suggests+receptor+interaction+or+competition.">Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition.</a> Genetics. 163 (1), 115-132 (2003).</li><li>Morley, J. F., Brignull, H. R., Weyers, J. J., Morimoto, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+threshold+for+polyglutamine-expansion+protein+aggregation+and+cellular+toxicity+is+dynamic+and+influenced+by+aging+in+Caenorhabditis+elegans.">The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 10417-10422 (2002).</li></ol>

我们什么都没有透露。

线虫同步几个漂白解决方案已被描述。我们尝试不同的食谱（ 见表一 ），在我们的手中，他们没有表现出显着性差异在蠕虫人口同步（ 图1）。然而，我们的实验也表明，参数，如温度（ 图2），比漂白液:虫量（ 图3）和M9的体积与胚胎培养孵化（ 图4）做影响蠕虫的生存与文化的适当通风。在我们的晃动的条件，而在一管15毫升1毫升的量，允许所有的蠕虫病毒的生存，5毫升的体积已经是太多了，让适当的卵孵化和可比到最大音量的15毫升（未显示） 。 线虫发展在发展过程中， <em&gt; C。线虫经过四个幼虫期（ 图5）成人阶段之前。胚芽行是一个很好的指标C.发展阶段线虫 。最简单的功能的C. Nomarski光学下，可以观察线虫的发展是外阴的发展，在L4早期阶段开始形成。起初，只有一小管腔观察，后来扩大到所谓的"圣诞树"形状，L4月中下旬。最后，通过对L4年底的外阴关闭（ 图6）。另一方面，DAPI染色可以观察性腺的发展。从四个单元在L1，L2和L3的细胞分裂和伸长性腺。在L3远端根尖细胞可以观察到，开始迁移背侧。减数分裂也开始由三级年底。在L4远端根尖细胞，达到他们的确切位置和生殖细胞分化成精子。结束最终由L4生产精子和卵母细胞开始生产。在adulţ蠕虫胚胎可以观察到子宫内（ 图7）。 发展和温度 C. elegans的发展在不同的速度取决于温度，而需要从鸡蛋奠定时刻约90小时，直到新的蠕虫病毒开始奠定自己的卵子在15℃，45小时有足够的时长大于25° ℃（ 图6）。设立条件和进行实验，研究在不同温度下的发展速度差，导致相对的灵活性。此外，它提供的可能性，不仅要监测一个特别的待遇或变更（例如温度敏感等位基因）的影响，而且还建立在开展一项特别实验的最佳条件。

basic caenorhabditis elegans methods: synchronization and observation

到线虫的分子和发育生物学的研究，在七十年代初开始，由悉尼·布雷，它已被广泛用来作为模式生物1。C。线虫具有关键属性，如简单，透明和生命周期短，都使得它一个合适的2多年的基本生物学研究的实验系统。这种线虫的发现具有广泛的影响，因为许多细胞和分子过程控制动物发展进化保守。 通过胚胎阶段和四个幼虫进入前动物达到成年线虫的生命周期。发展可以采取2至4天，根据温度。在每一个阶段，可以观察到几个特征性状。其完整的细胞谱系知识4,5与深annotat的共同其基因组的离子变成一个领域模型这种线虫老化7,8神经生物学，干细胞生物学9和生殖细胞生物学10多元。 一个额外的功能，使三线虫一个有吸引力的工作模式是获得人口在一个特定的阶段同步蠕虫通过一个相对简单的协议的可能性。易于维护和传播这种线虫添加到同步的可能性提供一个功能强大的工具来获得大量的蠕虫病毒，它可以RNAi屏幕，芯片，大规模测序，如小或高通量实验的各种使用，免疫或原位杂交，等等。 由于其透明度，C。线虫结构可以区分使用微分干涉对比显微镜显微镜下，也Nomarski显微镜复制。利用DNA荧光的DAPI（4&#39;,6-diamidino-2-苯基）粘结剂，例如，可能会导致个别细胞的识别和定位，以及与他们相关的亚细胞结构/缺陷。

Watch this Scientific Journal Video about Basic Caenorhabditis elegans Methods: Synchronization and Observation at JoVE.com

Basic Caenorhabditis elegans Methods: Synchronization and Observation

维护和线虫传播的难易<em&gt; C。线虫</em&gt;使一个很好的模式生物工作。同步蠕虫病毒的可能性，使相当数量的科目，在同一发育阶段的工作，促进一个特定的进程，在许多动物的研究。

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 ...

basic-caenorhabditis-elegans-methods-synchronization-and-observation

Nanyang Technological University

Research

JoVE Journal

Biology

Basic Caenorhabditis elegans Methods: Synchronization and Observation

46.1K 次观看.  Bellvitge Institute for Biomedical Research. 维护和线虫传播的难易 C。线虫使一个很好的模式生物工作。同步蠕虫病毒的可能性，使相当数量的科目，在同一发育阶段的工作，促进一个特定的进程，在许多动物的研究。

视频: Basic Caenorhabditis elegans Methods: Synchronization and Observation

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 ...

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 ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

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, ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

方法研究线虫线虫中固有的蛋白质聚集与年龄的变化

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

线虫的核苷酸和蛋白质提取联合

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...

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 ...