我们感谢Chris Webster对于执行图代表结果的3和Jason Watts和克里斯·韦伯斯特所示的初步实验对稿件有用的意见。资助这项研究是通过提供赠款来自美国国立卫生研究院（美国）（R01DK074114）到仲量行。在这项工作中所使用的一些线虫菌株是由线虫遗传学中心，这是由研究基础设施计划的美国国立卫生研究院办公室（P40 OD010440）提供资助。

多不饱和脂肪酸是对热，光和氧敏感。因此，必须制备脂肪酸补充剂板时，应考虑这样的脂肪酸是不暴露在过量的热和光。含有0.1％的Tergitol（NP-40）NGM介质被高压灭菌和部分冷却，之后，脂肪酸钠盐中加入并不断搅拌。所述板被允许在黑暗中进行干燥。脂肪酸由C摄取线虫培养于这些板然后可以通过气相色谱法来监测。 1。脂肪酸补充培养基的制备<ol><li>测量出的媒体组成部分成适当大小的烧瓶中。每1升中，添加17克细菌用琼脂2.5克胰蛋白胨，3克氯化钠，加入1ml 5毫克/毫升胆固醇溶解在乙醇和10ml 10％的Tergitol溶解在水中。 </li><li>加入80％的微孔水最终所需量和高压灭菌媒体以及空玻璃瓶（相当于不同的数脂肪酸浓度进行测试），以及适当的量筒。 </li><li>在水浴中设定为55℃，阴凉媒体当介质被冷却制备由破开的玻璃小瓶中，使用的安全防范措施，并确保玻璃粒子不混在的脂肪酸，脂肪酸的钠盐的工作原液。称出足够的脂肪酸，使一个100毫米的工作股票。 </li><li>带来的脂肪酸溶液至100mM的用纯化水的最终浓度。完全溶解的脂肪酸的钠盐通常需要约20-30分钟。吹扫的脂肪酸用氩气或氮气的工作原液，由于用惰性气体接触防止氧化。盖上瓶盖和存储作业储备，在黑暗中，直到媒体已经冷却。 </li><li> （可选）如果RNAi的媒体需要，测量氨苄青霉素和异丙基-β-D-1-硫代半乳糖苷（IPTG）解决方案在这里。 </li><li>当琼脂冷却到55°C每增加1升：1毫升1M MgSO 4干燥，将1ml的1M的CaCl 2和25ml的磷酸盐缓冲液（108.3克KH 2 PO 4和35.6克K 2 HPO 4，每1升高压灭菌）。添加过滤除菌氨苄青霉素和IPTG的解决方案，如果做的RNAi板。对于所有类型的板，加无菌水至使最终的介质体积至1L。 </li><li>附近的火焰，转移媒体的蒸压和适当大小的量筒，然后加入温水无菌水到所需体积。传输媒体回到初始瓶中，搅拌混合。 </li><li>靠近火焰，传输介质分成若干瓶等于浓度通过用蒸压测量被测试的数量和适当大小的带刻度的量筒。使用水浴直至脂肪酸工作股价已充分溶解维持等分媒体称为液体。 </li><li>将一个瓶子上的轰动板和搅拌，直到媒体是温暖的触感，但不热。请务必留下自己足够时间搅拌中的脂肪酸原液和倒片介质开始凝固之前。如果搅拌盘具有温度控制，将其设置为55°C。 </li><li>稀释100毫米脂肪酸的工作股票进入媒体所需的终浓度。搅拌介质，直至白色沉淀物是在溶液中（约1分钟）。媒体可能还是稍微浑浊之后。 </li><li> （可选）如果RNAi的媒体需要加氨苄青霉素至0.1毫克/毫升和IPTG至最终浓度为2mM。 </li><li>使用自动吸管援助和无菌25毫升吸管，加入8毫升媒体每60毫米板或每100mm板25毫升媒体倒媒体。重复脂肪酸加成和浇注板的其余瓶子的步骤。 </li><li>之后，琼脂凝固，覆盖脂肪酸补充板与框或在室温下储存在通风良好的抽屉从光氧化保护。 </li><li> E.种子大肠杆菌 OP50到平板平板干燥后的两天后。60 MM平板上，移液器300μl的OP50过夜培养（在37℃生长）的。如果RNAi的板倾倒，种子用300μlRNA干扰细菌后平板干燥后四天。板的干燥时间可能需要由于各种实验室环境中湿度的差异进行调整。 </li><li>孵育在黑暗环境中的板在室温下，而菌苔被干燥。 </li></ol> 2。由DGLA的补充诱导生精细胞破坏<ol><li> 线虫生长在含0.3 mM的DGLA（20时03 N-6）板块成为不育是由于生殖细胞在这两个幼虫阶段和成年线虫的破坏。 <ol><li>通过治疗妊娠雌雄同体用碱性次氯酸钠溶液准备L1幼虫的同步人口（对于一个10 ml的溶液：0.5 5M的NaOH，2.5毫升家用漂白剂，和7ml H 2 O的毫升）。轻轻摇动在此解决方案的妊娠直到雌雄同体成虫s溶解。鸡蛋将被保留，并且可以通过低速离心沉淀。 </li><li>在M9的缓冲液洗蛋准备3次（3克KH 2 PO 4，6克的Na 2 HPO 4，5克氯化钠，加入1ml 1米硫酸镁4，H 2 O至1 L。添加硫酸镁高压灭菌后）。以提供足够的氧气，重悬在鸡蛋15毫升的塑料管以5毫升M9缓冲液和岩石的终体积在摇床上过夜。 </li><li> L1幼虫应电镀的RNAi菌后用OP50，或一天播种后两天加入到补充板。蠕虫孵育在20℃，3天或直到他们达到成年阶段。 </li></ol></li><li>成虫可以拿下的使用解剖显微镜用足够高的倍率进行可视化的胚胎在子宫内发展不育。成功的生精细胞丢失会出现鸡蛋清子宫无效。 </li><li>蠕虫还可以砍下通过固定并与核酸染色CID染料diamindinophenylindole吲哚（DAPI），这有利于晶核在生殖细胞中的可视化和发育中的胚胎。一种快速DAPI染色是由采摘蠕虫成M9的缓冲区上表面玻璃9的下降来实现。 <ol><li>洪水手表玻璃用1毫升0.2纳克/立方米。 DAPI在95％的乙醇溶液，让坐在约5分钟。 </li><li>挑虫进入VECTASHIELD的幻灯片上的下降，然后盖上盖玻片。 </li><li>密封盖玻片，并储存于4℃在黑暗中过夜，以获得最佳的染色，但是滑动也可立即观看。得分为存在或不存在使用装有紫外线灯和过滤器的荧光显微镜生殖细胞的细胞核。 </li></ol></li></ol> 3。确认脂肪酸的摄取气相色谱法总体脂肪酸C的组成线虫可以通过生成脂肪酸甲酯（脂肪酸甲酯），它们然后被分离并用气相字符定量测定omatography 4。 <ol><li>洗它们了与水板，并转移到硅烷化13毫米×100毫米的玻璃螺丝顶管收集500-1000成虫。 </li><li>让蠕虫清偿重力，然后删除尽可能多的水可能用玻璃巴斯德吸管。 </li><li>用清水洗一次虫，然后再删除尽可能多的水可能。 </li><li>脂肪酸甲酯是通过加入1ml 2.5％的H 2 SO 4的甲醇溶液，然后加热，在70℃进行1小时的水浴中形成的。 </li><li>从水浴中取出试管并冷却1分钟。 </li><li>通过加入1.5毫升的水和0.25毫升己烷提取脂肪酸甲酯。 </li><li>回顾管和剧烈震荡。 </li><li>离心管中，在台式临床离心1分钟以最大速度己烷从含水溶剂中分离出来。 </li><li>转让正己烷（顶层）的GC样品瓶插件内的GC样品瓶，小心不要将任何水相。对于GC一nalysis，1-2μl，以正己烷脂肪酸甲酯被注入到适合脂肪酸甲酯分析极性毛细管气相色谱柱。在Agilent 7890 GC进样口被设定在250℃，用1.4毫升/分钟的流速，并在GC烘箱编程为在130℃的初始温度，这是保持1分钟。随后，将温度斜坡10℃/分钟直到190℃，然后再次升温速率5℃/分钟直到210℃并保持另外1分钟。 </li><li>以确保已发生的DGLA的那摄取，用火焰离子化检测器（FID）或质谱（MS）检测，用真实标准为C的鉴定分析脂肪酸甲酯线虫脂肪酸。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

这里我们描述的补充C的方法线虫与食物中的不饱和脂肪酸。如上所述，必须注意在多不饱和脂肪酸补充板的准备，因为在多不饱和脂肪酸的双键的反应自然会导致这些不饱和脂肪酸，通过热和光11是氧化敏感。为了避免氧化，该多不饱和脂肪酸加入到液体琼脂培养基后，媒体已经冷却至55℃，并存储板在黑暗环境中是很重要的。 他人已经介绍的游离脂肪?...

脂肪酸是膜的必要的结构部件，以及有效的能量储存分子。此外，脂肪酸可以从细胞膜裂解通过脂肪酶和酶促修饰以产生信号的效应1。天然存在的多不饱和脂肪酸（PUFA），含有两个或多个顺式双键。 ω-3脂肪酸和ω-6脂肪酸是彼此区分的基础上的双键相对于脂肪酸的甲基端的位置。健康饮食需要两个ω-6和ω-3脂肪酸。然而，西方的饮食是特别丰富的omega-6脂肪酸，差ω-3脂肪酸。高ω-6与ω-3脂肪酸的比例是与心血管和炎性疾病的风险增加，但是，具体的脂肪酸的精确有益和有害功能都不能很好地理解2相关联。蛔虫秀丽隐杆线虫elega因为它综合了所有必要的酶来生产一系列的ω-6和ω-3脂肪酸，包括ω-3去饱和酶的活动，是在缺席的大多数动物3,4纳秒是研究脂肪酸的功能是有用的。突变体缺乏脂肪酸去饱和酶的酶不能产生特定的PUFA，从而导致一系列的发育和神经缺陷4-6。 探讨膳食脂肪酸的生理效应，我们已经开发出一种生化检测与遗传分析兼容同时使用突变和RNAi敲除技术在C线虫 。补充特定的多不饱和脂肪酸是通过将脂肪酸的钠盐溶液的琼脂培养基之前浇注来实现。这将导致在多不饱和脂肪酸的摄取由大肠杆菌大肠杆菌的食物来源，它积聚在细菌膜。C.线虫摄取的含PUFA的细菌，这饮食补充足够的抢救有缺陷的多不饱和脂肪酸缺乏的突变体TS。大多数脂肪酸补充有对野生型动物没有有害的影响，但是，具体的ω-6脂肪酸，尤其是二高-γ-亚麻酸（DGLA，20点03的n-6）引起℃的永久性破坏线虫生殖细胞7,8。 气相色谱法是用于监视补充脂肪酸的细菌食物源（OP50或HT115）以及线虫的吸收。在加入洗涤剂的Tergitol（NP-40）中的媒体的允许通过整个板和的脂肪酸由大肠杆菌更高效摄取均匀分布的脂肪酸大肠杆菌和线虫。我们已经发现，不饱和脂肪酸可以容易地采取了由细菌和C。线虫 ，但是饱和脂肪酸的摄取是非常低效率的。本文将描述一步一步如何与脂肪酸补充琼脂培养基，以及如何监测在日脂肪酸的摄取Ë线虫用气相色谱法。

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

脂肪酸是必需的许多细胞功能。他们作为高效的储能分子，补膜的疏水核心，并参与各种信号通路。 秀丽隐杆线虫综合了所有必要的酶来生产一系列的ω-6和ω-3脂肪酸。此，结合了简单的解剖学和可遗传工具的范围，使之成为有吸引力的模型来研究脂肪酸功能。为了研究介导的膳食脂肪酸的生理作用的遗传途径，我们已开发出一种方法，以补充C。线虫的饮食与不饱和脂肪酸。补充剂是改变蠕虫的脂肪酸组合物，一种有效的手段，也可用于抢救中脂肪酸的缺失突变体的缺陷。我们的方法用线虫生长培养基琼脂（NGM），补充有脂肪acidsodium盐。在补充板的脂肪酸成为incorpora特德进入细菌的食物来源，然后采取了由C的膜线虫饲料的补充细菌。我们还描述了一种气相色谱协议来监控发生在补充蠕虫中脂肪酸组成的变化。这是为了补充C的大和小群体的饮食的有效方法线虫 ，允许的范围内对本方法的应用。

在C的补充线虫饮食是由细菌食物源的摄取和结合脂肪酸到细菌膜的能力的限制。要确定大肠杆菌的能力大肠杆菌 OP50吸收各种脂肪酸进入其的膜，OP50被镀到无补充剂，0.1毫米和硬脂酸（18:0），油酸钠（18:1 N-9），和钠DGLA（20 0.3mM的浓度媒体：3N-6）。平板在室温下干燥在黑暗中3天，并保温在20℃下进行3天。细菌草坪被轻轻地刮草坪成水与火焰消毒压舌板收集。细菌通过?...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

的&lt;em&gt;线虫实验&lt;/ em&gt;的是一个有用的模型，以探讨发展和生理学的多不饱和脂肪酸的功能。这个协议描述补充的&lt;em&gt; C的一种有效的方法线虫&lt;/ em&gt;的饮食与多不饱和脂肪酸。

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K 次观看.  Washington State University. 的线虫实验的是一个有用的模型，以探讨发展和生理学的多不饱和脂肪酸的功能。这个协议描述补充的 C的一种有效的方法线虫的饮食与多不饱和脂肪酸。 

视频: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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 ...

科研

生物学

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 ...

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 ...

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, ...

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 ...

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 ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

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

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

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

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 ...