我们想感谢迈克尔Kacergis，为技术援助和联丰吴的讨论和阅读的手稿。这项工作是支持的职业生涯发展奖由美国国立卫生研究院/ NIDDK（K08DK087941到AAS），NIH / NIDDK训练的授予（5T32DK007028到ECP），埃里森医学基金会的新学术在老化奖（AAS），和查尔斯Ĥ胡德基金会儿童健康研究奖（AAS）。

1。矩形安培/ TET（A / T）的LB琼脂平板和NGM的IPTG RNA干扰板的制备应制备的A / T板前1-4周，使用并储存在黑暗中在4℃下<ol><li>对于1 L的媒体加到1升的去离子水，32克的LB琼脂，1.5毫升2 M的NaOH，和2克Bacto琼脂。液循环高压灭菌40分钟。后冷却至60℃，加入3毫升四环素和0.5 ml氨苄青霉素。倒入45毫升的媒体，每块板。 </li></ol> 96 RNA干扰板准备提前3天至2周。 <ol start="2"><li>要倒入25 96孔的RNAi板，准备1 L的线虫的生长介质（NGM）：1升的去离子水，3克氯化钠，17克琼脂糖，和2.5克细菌蛋白胨。高压釜液循环，在121°C为40分钟，搅拌棒。放凉至60°C，搅拌热板集</li><li>当冷却至60℃，加入以下股票的解决方案：1毫升1米硫酸镁 ，1毫升5毫克/毫升的胆固醇，25毫升1 M KH 2 </suB̶：&gt; PO 4（pH 6.0）中，1毫升的1M的CaCl 2，1毫升200毫克/毫升的羧苄青霉素，和5ml 1M的IPTG（见材料表食谱）。 </li><li>分送平底96孔板中使用电子的多通道移液器到每个孔中加入150μl的RNAi媒体。避免气泡。保持介质浸渍在60℃的水浴中，同时分配在一个无菌的不锈钢容器。 </li><li>保持RNA干扰板的水平，直到琼脂糖巩固。板可以倾长达2周的提前，并储存在4℃下</li></ol>第1天 2。邮票冷冻库板到矩形安培/ TET（A / T）的LB琼脂平板上<ol><li> A / T牌暖到室温，让他们在黑暗中。 </li><li>转移96 - 孔的RNAi库板从-80℃干冰。附近的本生灯打开密封铝盘。 </li><li>杀菌96针复制器串联浸渍销（每次10秒），在10％的漂白剂，去离子H 2 O，和70％E“TOH随后通过引脚通过一个火焰。重复乙醇和火焰步骤。 </li><li>应用复制到冷冻96的RNAi甘油，确保每个引脚接触面每口井。 </li><li> A / T板，邮票上画一个小圆圈，每个引脚不破坏琼脂表面。 </li><li>再密封和返回图书馆板至-80℃。孵育加盖A / T板在37°C过夜细菌的生长。 </li></ol>第2天 3。长大RNA干扰的细菌克隆隔夜在深井块<ol><li> 37°C和A / T牌，确认细菌的生长。 </li><li>填充一个96孔的深阱块的每个孔中，用1.5毫升用200微克/毫升的羧苄青霉素的LB。 </li><li>消毒96针复制（见2.3）。 </li><li>应用复制到A / T板的顶部，确保引脚接触各种细菌的RNA干扰克隆。提起复制到深井块，浸引脚。漩涡，慢慢松开，使CErtain不污染邻井。 </li><li>呼吸容易膜密封块。孵育过夜，在37℃下搅拌（​​950 rpm离心在MT Infors Microtiteron II摇床，优选的，或250 rpm在一个25毫米掷摇床中）。 </li></ol>第3天 4。种子的RNAi细菌无性系到96孔NGM的RNAi板上<ol><li>删除96的RNAi板4°C温至室温。 </li><li>删除深井块，并在4000×g离心10 min。 </li><li>快速反转块成片漏媒体的，并加盖倒到纸巾上，以消除多余的介质。 </li><li>在层流罩下，添加40微升的LB的各孔和密封用无菌膜用200微克/毫升的羧苄青霉素。 </li><li>返回块至室温细菌摇床10分钟，再悬浮的颗粒，或手动重新悬浮通过小心地移液粒料。 </li><li>在层流罩，取出无菌薄膜和转移细菌深到96孔的RNAi板以及块。加入40μl再悬浮细菌行数&#39;A&#39;和&#39;H&#39;，和35μl从所有其他行。更多的液体被加入到边缘的孔中的96 - 孔的RNAi板，以防止过分干燥和开裂的琼脂糖。 </li><li>保留4-6小时晾干罩板发现，检查定期为干燥。干菌呈透明的，不混浊。盖，反转板，室温孵育过夜。 </li></ol> 5。准备同步人口的蠕虫鸡蛋准备每6 96的RNAi板的用于两个10公分板充满了许多怀孕的蠕虫。请务必蠕虫的非饥饿状态至​​少两代（7天）。 <ol><li>准备一个同步人口L1如前所述的20。对于20毫升的漂白剂溶液，使用的NaOH（10 M）1毫升5毫升去离子H 2 O，和10毫升M9的缓冲液4毫升漂白剂，。 </li><li>过夜同步蠕虫在10ml M9在一个15毫升无菌聚丙烯管中在20℃下与旋转</li></ol>第4天 6。蠕虫的RNAi板<ol><li>以3,000×g离心1分钟，离心蛋税号。吸出上清液，远离L1刚孵化的颗粒。 5毫升M9的缓冲液重悬在0.0005％的Triton X-100的。添加少量的洗涤剂防止蠕虫粘到吸移管和没有对脂​​肪染色效果。 </li><li>显微镜下计数蠕虫。如果有必要，调整浓度每微升至10-15蠕虫。 </li><li>在层流罩中，为每个96孔板中接种，成一排的一个浅孔分析块的各孔中加入75微升的重新悬浮的蠕虫。使用手动多道移液器，添加50-75蠕虫于5μl到各孔，96孔的RNAi板。 </li><li>允许板，在通风橱中进行30〜60分钟的干燥。干燥时，在显微镜下，蠕虫病毒应该会出现爬行，不抖动。 </li><li>成长蠕虫病毒的RNAi板在20°C〜64小时。 </li></ol> 7天 7。修复，并在尼罗红染色蠕虫<ol><li>从孵化器中删除的RNAi板。在显微镜下检查和记录饿死或抖动（湿）井排除进一步的分析，这些条件会影响脂肪的质量。 </li><li>使用的电子重复移液器，添加150微升PBS，0.01％的Triton X-100的到的RNAi板的各孔中。一个手动多道移液器，结构和传输液体的蠕虫深96孔PCR板中的相应行。避免破坏琼脂。共PBS，每孔300μL蠕虫在重复上述步骤。 </li><li>当四个板已经转移，板以500rpm离心1分钟。 </li><li>吸液上清液，使用96针的真空抽吸器，在井的底部离开〜25微升的液体和蠕虫颗粒。 </li><li>添加150μl的40％异丙醇的各孔固定动物。足够高的速度肯定的是，移液器混合蠕虫沉淀用40％的异丙醇。在室温下孵育3分钟。 </li><li>离心机板揭露，持续1分钟，在500 rpm的转速下。 </li><li>吸弃上清液，用96针吸气器或手动，只留下25微升40％的异丙醇+蠕虫在井的底部沉淀。 </li><li>向每孔加入150μl的尼罗红染色溶液在使用前立即制备：6微升尼罗红的0.5毫克/毫升在丙酮中每1毫升的40％的异丙醇的原液。 </li><li>密封板与非无菌粘接膜和至少2小时，在黑暗中染色。 </li></ol> 8。洗净，图片蠕虫在高通量显微镜<ol><li>为1分钟，在500 rpm的转速下离心和吸但25μl的尼罗红染料。 </li><li>加入150μl的PBS，0.01％的Triton X-100的各孔中，并存储在至少30分钟的暗。 </li><li>以500rpm离心1分钟。 </li><li>从每口井的底部有2个7微升快速使用12通道移液器，吸动物冲程和分配到一个96孔的聚四氟乙烯印刷载玻片上。小心，不删除蠕虫，从每口井的幻灯片中删除9.5μl的PBS。如果你的玻璃的幻灯片不适合直接在显微镜安装，自定义加工铝滑轨持有人必须被用来发动蠕虫。 </li><li>慢慢的下盖滑到96幻灯片。这个步骤可能需要一些做法，以避免气泡或跨越到其他井的蠕虫病毒。用粘合剂粘性的安全角落。 </li><li>明场和荧光与GFP / FITC滤光片自动显微镜的图像幻灯片。脂质信号的光漂白剂容易，所以它应该有系统地成像在所有的井，手动，，作为漂白会导致人工井之间的差异。分析的详细信息，请参阅代表性成果和讨论。 </li></ol> 9。生化血脂测定采用固相萃取（SPE）和气相色谱 - 质谱法（GC / MS） <p class=“jove_content”&gt;验证尼罗红荧光血脂水平的基础上，然后通过质谱分析（GC / MS），气相色谱法，可以执行的脂肪酸甲基酯（FAME），甘油三酯（TAG）和磷脂（PL）从来自蠕虫病毒颗粒。涉及有机物的步骤应在通风橱中进行。应小心，脂质保持避光在氮气氛下，在延长的贮存或孵育步骤，因为它们是非常容易氧化。蜗轮从2,500-5,000同步动物粒料被用作输入，完全相同的，除了上述制备动物生长于10厘米的RNAi板。 <ol><li>洗净2,500-5,000蠕虫从每个10厘米板用10ml M9缓冲液到15毫升锥形聚丙烯管。佩莱蠕虫在500×g离心一分钟，并再次用10ml M9的缓冲液洗沉淀。佩莱在500×g的动物。吸干离开250微升总体积，和任一冷冻液氮并存储在-80℃下，在氮气氛下FOŕ后处理或直接进入。 </li><li>该蠕虫病毒颗粒可采取直接步骤9.3，如果甘油三酯质量是被归到磷脂质量8,13,21。然而，如果研究者希望正常化蛋白质含量或DNA含量，蜗杆沉淀应超声处理10分钟，30秒开，30秒关，在4℃下的浴中的强度最高的超声波发生器上如果蛋白质或DNA正常化的需要，取出5微升等份，并用Bradford试剂或相似或总DNA含量柱净化后，用紫外分光光度计测定总蛋白浓度。 </li><li>加入到1.5 ml 2:1的氯仿：甲醇在一个线程的高硼硅玻璃管的蠕虫。所有的有机溶剂，应使用玻璃吸管转移。 </li><li>使用wiretrol 50微升吸，加25海里磷脂在50μl的标准，和16.7 nm的甘油三酯在50μl的标准。这两种标准都应该溶于2:1的氯仿：甲醇，盖紧，并储存在氮气氛下从光在一个-80℃的冰箱中，以避免氧化保护。 </li><li>酚醛帽帽和涡。提取物在室温下1小时，每15分钟涡旋式。 </li><li>以1,000 rpm离心5分钟。没有尸体的下层有机相转移到文化硼硅管。 </li><li>加0.3毫升0.9％NaCl溶液中，旋涡和离心机在1,000 rpm离心5分钟。 </li><li>底部有机层转移到一个新的硼硅酸盐的培养管中，确保不超过任何的水相转移。 </li><li>在氮气氛下的干燥有机相。首先固相萃取（SPE）重悬干脂质在1毫升氯仿。另外，对于详细不同的脂质类，包括磷脂类，糖脂，鞘脂，甘油二酯，甘油三酯，游离脂肪酸，和胆甾醇酯的分离和分析，薄层色谱法，率先在 C由瓦特实验室线虫 22可以用于代替的SPE。血脂可也是BË分离高效液相色谱法和组分分析GCMS。也可用于复杂的方法的的全lipidome剖析者liquid-chromatography/MS（LCMS）23-26。 </li><li>组装硅胶固相萃取柱SPE歧管。预平衡的柱用3毫升氯仿。允许溶剂通过重力流。 </li><li>脂质样品加载到SPE柱，收集流过作为第一馏分（TAG分数）的一部分，在一个带螺纹的硼硅管。大多数中性脂肪会通过在这第1毫升流过。加入3毫升氯仿，充分洗脱TAG部分，收集流过的TAG部分管。 </li><li>删除和CAP的第一个集合管，并用干净的收集管更换。用5毫升9:1的丙酮：甲醇洗脱鞘糖脂。但是，我们并没有惯常分析该馏分的脂肪酸组合物，它可能包含有价值的信息，并且可以保存用于制备甲基酯和免费的鞘氨醇碱作进一步的分析利用如先前所述的TAG和PL 27的不同的专门程序。 </li><li>更换的第二螺纹高硼硅收集管的糖脂收集管。用3毫升甲醇（PL馏分）洗脱磷脂。 </li><li>馏分可以被存储在-80℃下在这一点上在氮气下盖紧。干纯化的氮气氛下的脂质。为了加快干燥，在干燥的加热块在33°C。绝不会储存在干燥的形式，因为它们是在干燥的状态下特别是易被氧化的脂类。 </li><li>重悬干燥的脂质成分在2毫升2％H 2 SO 4在甲醇涡旋和孵育过夜，在55℃水浴创造脂肪酸甲酯盖紧。必须小心，绝对没有得到的FAME反应中的水，这将极大地抑制脂质衍生。我们已经找到更有效的衍生过夜培养和文献表明，减少脂质过氧化反应在较低温度下发生杜里吴FAME准备28。的另一种更快速的方法是使用在甲醇中的2毫升2.5％H 2 SO 4和衍生化一小时，在70或80℃21,22,29。 </li><li>第二天早上，从浴中删除，并允许冷却。添加1.5毫升H 2 O（以淬灭反应），然后0.3毫升己烷（提取的脂肪酸甲酯）到每个文件TAG和PL馏分。用力摇晃，并在1000转离心5分钟。 </li><li>非常小心地取下顶部正己烷层使用标准的移液器或巴斯德吸管。分装到自动进样器管。请一定不包含任何酸化甲醇，因为这会破坏气相色谱柱和MSD。 </li><li>到GC / MS帽和负载样品。将样品传送到微插入安捷伦封端的PTFE-硅氧烷PTFE螺丝帽的小瓶在己烷中。它被转移到安捷伦的GCMS模型6890/5973N配备Supelcowax-10（24079）30米，250微米的石英毛细管柱。一微升注入诠释办公自动化不分流进样口设置在250℃和13.33 PSI超纯氦气使用的载气。该协议运行在一个恒定的1毫升每分钟如下： </li></ol><table border="1"><tbody><tr><td> 步骤 </td><td> 指令 </td><td> 目标温度 </td></tr><tr><td> 1 </td><td>保持2分钟</td><td> 150°C </td></tr><tr><td> 2 </td><td>斜10°C每分钟</td><td> 200°C </td></tr><tr><td> 3 </td><td>保持4分钟</td><td> 200°C </td></tr><tr><td> 4 </td><td>斜5°C每分钟</td><td> 240°C </td></tr><tr><td> 5 </td><td>保持3分钟</td><td> 240°C </td></tr><tr><td> 6 </td><td>斜10°C每分钟</td><td> 270°C </td></tr><tr><td> 7 </td><td>保持5分钟</td><td> 270°C </td></tr></tbody></table>溶剂延迟3分钟，在MSD使用的过滤，以最大限度地减少磨损阿曼特。此后，在50和550之间道尔顿群众与MS四倍集检测在150℃下和ms的源设置在230℃，与热辅助温度为270℃。

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作者有没有利益冲突披露。

该协议允许进行快速，大规模筛选C.血脂水平线虫 ，它可以很容易地适应高吞吐量屏幕。与以前使用的方法，它涉及一个漫长的一步，多聚甲醛固定的几个冻融循环8,10，我们的协议需要一个简单的3分钟固定在异丙醇。我们已经发现，通过染色C.线虫在40％异丙醇，它们成为可自由可渗透的尼罗红（Nile red），而不使用额外的冻结-解冻或固定步骤。此外，尼罗红（Nile red）选择性荧光在绿色光谱18,19,31疏水隔室的，没有脱色是必要的。总体而言，可以采取动物，从RNA干扰板的图像动物染色，并准备在刚刚超过2.5小时。此方法可以执行相对廉价且具有高的再现性。该方法不依赖于C 的能力线虫吸收或浓缩的染料，并因此不具有供给为基础的方法，用活体染料相同的局限性。由于测量的再现性和精度的方法，该方法是适合于通过RNAi筛选大量的基因失活。血脂水平的定量所需的尼罗红染色后固色剂，我们建议使用4-6次生物学重复与适当的控制和统计测试应用。 这是可能的，很多小动物的基因失活，导致由于RNAi技术克隆造成延误或逮捕发展出来的脂肪监管机构的屏幕，无论任何连接到脂肪代谢。虽然图像分析程序中，我们通过标准化的荧光强度的蠕虫，利用不同大小的蠕虫占个别研究人员也可能要考虑比较相似的发展阶段的小动物，野生动物的脂肪染色水平。在具有明显的低脂肪量小或发育拘捕的动物的情况下，我们认为没有单一的模式是足以证明脂质代谢的改变。几个后续实验，包括SPE-GCMS，必须确定这些基因的的脂质调节功能的。发育匹配N2 L2控制动物相比，对于已知的代谢调节FASN-1，SPE-GCMS分析是必要的，以确认明显的低脂肪的表型发现成人对照动物相比时。虽然尼罗红脂肪染色显示低脂肪含量相对RNA干扰技术，控制成年阶段相匹配的L2控制RNA干扰动物相比，没有明显的差异可见一斑。因此，使用第二个方法， 即生化定量的TAG / PL比是必要的，以确定一个FASN-1的是一个真正的调节器的脂肪量，FASN-1 RNAi是生化区别于阶段匹配L2控制的RNAi动物。 20或常规尺寸的聚四氟乙烯印刷8至12孔显微镜载玻片。唯一的限制是，迅速尼罗红染色，光漂白剂的脂滴的绿色荧光，系统化，需要使用统一的曝光条件，以确保图像之间的可比性。我们发现，一个完全自动化的显微镜具有均匀的图像采集条件下效果最好这种效果。 本协议的一大优点是图像收集后，他们可能会被保存为未来的再分析。可以使用相同的图像，来确定车身尺寸除了脂肪含量。此外，由于计算程序变得更加先进，有单蠕虫的前景分析，产生统计上的显著成效从一个单一的井。因此，我们的方法，利用高通量显微镜具有一定的优势出版的屏幕的方法，使用COPAS Biosort的量化荧光信号32,33。但是，这些方法是绝对免费的。 精确，生化测定脂肪含量的固相萃取和GC / MS证实了尼罗红染色的主要脂肪储存在 C 线虫 。虽然实现了从固定剂尼罗红染色和生化血脂测定的绝对定量，往往不完全匹配，这两种方法产生高重现性，在统计学上显着的结果，同意定性。此外，这种方法可以定制，以满足个别研究人员可能需要进一步分析的单独类别的糖脂，磷脂，甘油三酯的各种样品中存在的具体需求。应该指出的是，其他团体使用的技术THAŇ固相萃取分离脂质类之前GC / MS 22。薄层色谱（TLC）和高压液相色谱（HPLC）超过SPE具有优势的，因为它们可能会对能够更好地分离不同的中性脂肪（ 如 TAG，二酰基甘油，游离脂肪酸，和胆固醇酯）从极性脂质（磷脂酰胆碱，磷脂酰乙醇胺）和乙二醇鞘脂。选择SPE，因为它需要一个最低的起始原料（2500-5000蠕虫），它强调的主要长期能量储存，标签，从而弥补了大部分的中性脂质馏分4，速度快，无需专门的设备或板。应当强调的是，根据标准的混合物，SPE完全分离的TAG从PL的，并且是高度可重复性和可靠的（参见图5A）。虽然脂肪酸甲基酯的定量和分析需要的GC / MS，此设备是常用的，并且如果不是局部，样品可如上生成的，并存储在-80℃下在氮气氛下，直到分析的协作者或商业的GC / MS分析服务被配置。 的的GCMS输出包含上每个脂质种内各脂肪酸的高分辨率数据。这允许确定详细的样本之间的差异相比。作为一个例子，研究者可以决定是否被修改了某些脂肪酸的水平更显着的daf-2，则lpd-3或FASN-1的RNAi对矢量控制比其他丰富。因此，这个非常强大的工具可以提供详细的信息，改变脂质种的丰富的，任何实验操作。 在我们的分析中，我们最常TAG质量标准化PL质量。虽然蛋白质或DNA也可以用于从超声波处理的蠕虫颗粒的等分试样测定后正常化脂质质量，我们发现，这些方法可能会为int在所有的移液步骤，并在每次提取的样品回收率从roduced人为错误。这是因为这个原因，我们选择了我们的归一化因子，而不是使用PL质量。非自然标准都是通过固相萃取的所有步骤进行的，并在开始的协议（tritridecanoin为TAG，1,2-diheptadecanoyl-sn-甘油-3 -磷酸-（1&#39;-rac-甘油）用于PL）引入FAME准备，并保证标记和PL组分的定量和有代表性的恢复。不能作出同样的保证，如果调查使用蛋白或DNA正常化TAG质量。我们相信，PL是最强大的压力表比较TAG样本之间的水平，因为它已被先前的研究显示，PL只有微小的改变，相同的刺激，影响大的质量在TAG水平的变化， 例如扩展饥饿或增加运动34 - 36。 PL被认为扮演一个结构性的作用，保证了膜的流动性和蜂窝电话集成grity和信令的角色，而不是作为能量储存37-39。在我们的手中，的TAG / PL比最紧密的匹配是通过与尼罗红染色固色剂为基础的脂质，并同意与其他组21中发现的。一种替代的策略是使用由瓦特实验室，在那里的百分比计算的脂质文件TAG与总脂质9,22,29相比 。 使用非原生类型的TAG和PL标准，确保没有原生的脂肪酸将被纳入正常化计算。链长度的选择纯粹是根据这些标准的需要，以便不干扰的固有脂肪酸的质谱。我们已经发现，13:0和17:0脂肪酸提供最佳服务这一宗旨。这是可能的，给定的不同的化学性质的短链脂肪酸，我们不自然的脂肪酸标准可能不是代表恢复的所有的链的长度，用diff的脂肪酸或链条erent饱和指数。因此，它是可能的，在SPE或GCMS，我们可能会看到在纯化或脂​​质的不同链长的检测之间的效率差异。根据这和不同的电离电不同的脂肪酸中的GCMS，它是不能够进行绝对的陈述的任何给定的脂肪酸中的丰度。因此，我们只有一个单一的实验样本在任何给定的脂肪酸丰度之间的比较。如果一个人想绝对定量的脂肪酸，1的方式，这可以实现是将运行与上述加有已知量的稳定同位素标记的13 C版本，或类似的脂肪酸制备的样品，因此，它可能是根据质量MSD中的母体离子，例如区分。由于我们只比较相对量的脂质类或任何特定的脂肪酸，我们的13:0的TAG和17:0 PL标准，充分达到这个目的，在最低的成本，以每类脂质保证足够和有代表性的恢复。 直到此时，一直缺乏共识的解释，通过各种方法取得了一定的成绩，和现行的方法应该是什么。整体而言，这应该指出，没有一种方法在隔离非常适合研究C 中的脂肪代谢线虫 。然而，通过使用多个筛选和验证方式，可以实现对脂质调控基因上获得的数据的信心。因此，我们打算为我们的协议为基准，为今后的工作，在该领域的C.线虫脂肪的研究。

保护人类和线虫的代谢途径之间，使之成为一个强大的模式生物，研究肥胖。虽然C。线虫不具有专用像在哺乳动物脂肪储存的脂肪细胞，它们做店甘油三酯在脂滴1中，并具有许多相同的调节脂肪储存和能源使用2,3。为了测定C.脂肪含量线虫的几种方法已经提出了不同程度的易用性和成功。最近一次普遍使用的荧光灯，重要的染料4-7被质疑，这些试剂被证明是染色的不同C.主要的脂肪储存仓库的一个隔间线虫 1,8-11。非荧光染料固色剂为基础的，如苏丹黑油红-O，而强调脂滴的蠕虫病毒12-14取得成功，不会有大的动态范围，作为荧光定量百分之染料8,13,15。此外，荧光和非荧光染料固定电流的方法是费时和冻融涉及多个步骤和/或使用的有毒化学品1,9,10。使用特定BODIPY-标记的脂质类似物已报道当馈送到生活与短期标签15蠕虫时，以突出显示脂滴。然而，这依赖于染料的吸收，并因此由C.偏压的处理和BODIPY脂肪酸的代谢类似物。已建立的脂质染色的染料是无标记，相干反斯托克斯拉曼散射（CARS）或受激拉曼散射（SRS）显微镜，利用脂质分子的的特征振动特性的可视化脂肪储存10,16， 17。然而，此方法需要昂贵的专用设备是，充其量是低吞吐量。 为了满足需要快速，可扩展的ANALYsis文件的中性脂质店的C.线虫代谢的研究中，我们将介绍一种新型的，高固色剂为基础的尼罗红脂质染色重现性的方法。的亲脂性染料尼罗红光谱和物化性质诱导在其激发发光峰的黄色-金-光谱的移位，允许它只有当在富含脂质的环境中，但不是在极性较大的环境18中的绿色的发光光谱的荧光，19。因此，脂滴简单染色后，可以检测出所使用的绿色荧光蛋白（GFP）的过滤器设定为荧光显微镜。这项技术的易用性和可负担使得它非常适合于高通量筛选。我们还演示了配对的生化测定甘油三酯和磷脂固相萃取和气相色谱 - 质谱法，严格的方法。生化血脂测定与尼罗红为基础的微观测定C.的发吨的质量，并应作为与微观血脂测定结果的确认。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 96针复制</td><td> Nunc公司</td><td> 250520 </td><td></td></tr><tr><td> LB琼脂</td><td>美国生物</td><td> L1500 </td><td></td></tr><tr><td>琼脂糖</td><td> Invitrogen公司</td><td> 16500500 </td><td></td></tr><tr><td>机械移液器</td><td>百得M线</td><td> M10，M100，M300 </td><td></td></tr><tr><td>电子移液器</td><td>百得E行</td><td> E1200 </td><td></td></tr><tr><td> 1 M KH 2 PO 4的 </td><td>西格玛</td><td> P0662 </td><td>高压灭菌的pH为6.0 </td></tr><tr><td> 1 M 硫酸镁 </td><td>西格玛</td><td> M2643 </td><td>灭菌AUToclaving </td></tr><tr><td> 1 M 氯化钙 </td><td>西格玛</td><td> C2661 </td><td>高压灭菌</td></tr><tr><td>氯化钠</td><td>西格玛</td><td> S5886 </td><td></td></tr><tr><td> 5毫克/毫升的胆固醇</td><td>西格玛</td><td> C8667 </td><td>在100％乙醇中</td></tr><tr><td> 200毫克/毫升羧苄青霉素</td><td>美国生物</td><td> C1100 </td><td>过滤消毒0.2μ </td></tr><tr><td> 1 M IPTG </td><td>美国生物</td><td> I8500 </td><td>过滤消毒0.2μ </td></tr><tr><td> 100毫克/毫升氨苄青霉素</td><td></td><td></td><td>过滤消毒0.2μ </td></tr><tr><td> 5毫克/毫升四环素</td><td></td><td></td><td>在100％乙醇中</td></tr><tr><td> Bacto琼脂</td><td> BD </td><td> 214040 </td><td></td></tr><tr><td> 96孔TC板</td><td> BD-FALC上</td><td> 353072 </td><td>对于96的RNAi </td></tr><tr><td>矩形A / T牌</td><td> Thermo Scientific的</td><td> 242811 </td><td>对于冲压RNA干扰</td></tr><tr><td> LB媒体</td><td>美国生物</td><td> L1530 </td><td>按照制造商的指示编制</td></tr><tr><td> 96孔深孔含量块</td><td>康宁</td><td>深圳科诗特3960 </td><td>矩形V型底检测块</td></tr><tr><td> 96孔浅孔检测板</td><td>康宁</td><td>深圳科诗特3956 </td><td>圆形V型底试验块</td></tr><tr><td>无菌密封膜</td><td> VWR </td><td> 89134-432 </td><td></td></tr><tr><td>铝箔封口膜</td><td>康宁</td><td>深圳科诗特6570 </td><td>热电偶套管的密封胶带96 - 孔板</td></tr><tr><td> M9缓冲</td><td>看到了希望， 等。 </tD&gt; <td></td><td>配方下面， </td></tr><tr><td>的Triton X-100的</td><td> Bio-Rad公司</td><td> 161-0407 </td><td></td></tr><tr><td>尼罗红</td><td> Invitrogen公司</td><td> N-1142 </td><td></td></tr><tr><td>异丙醇</td><td>费舍尔</td><td> BP2632-4 </td><td></td></tr><tr><td> 96针吸气泵</td><td> VP科学</td><td> VP177A-1 </td><td></td></tr><tr><td> 96孔特富龙假面显微镜载玻片</td><td> Thermo Scientific的</td><td>习惯</td><td>还可以订购Trevigen 96 Cometslide </td></tr><tr><td> 96黄金密封Coverslides </td><td> Thermo Scientific的</td><td>习惯</td><td>也可以用第二个96 Cometslide </td></tr><tr><td>深孔PCR板</td><td> VWR </td><td> 89049-178 </td><td></td></tr><tr><td>非无菌粘合薄膜</td><td> VWR </td><td> 60941-070 </td><td></td></tr><tr><td>高通量显微镜</td><td>徕卡</td><td> DM6000全自动化的前96级</td><td> 49002 GFP与色度滤波器组和2个目标</td></tr><tr><td>浴超声波破碎</td><td> Diagenode </td><td> Bioruptor XL </td><td></td></tr><tr><td>氯仿</td><td>西格玛</td><td> 366927-4L </td><td></td></tr><tr><td>甲醇</td><td>费舍尔</td><td>越F456-4 </td><td></td></tr><tr><td>磷脂标准</td><td>阿凡提极性脂质</td><td> 830456P </td><td> 1,2-diheptadecanoyl-sn-甘油-3 -磷酸-（1&#39;-rac-甘油） </td></tr><tr><td>甘油三酯标准</td><td> NuChek准备</td><td> T-135 </td><td> tritridecanoin </td></tr><tr><td> Wiretrol 50μL移液管</td><td>费舍尔</td><td> 21-176-3A </td><td>德拉蒙德Scientfic </td></tr><tr><td>丙酮</td><td>西格玛</td><td> 320110-4L </td><td></td></tr><tr><td>硫酸</td><td>费舍尔</td><td> A300-500 </td><td></td></tr><tr><td>正己烷</td><td>费舍尔</td><td>越H306-1 </td><td></td></tr><tr><td> SPE硅胶柱</td><td> Thermo Scientific的</td><td> 60108-317 </td><td> Hypersep SI，100毫克树脂床</td></tr><tr><td>固相色谱法歧管</td><td>西格玛</td><td> 57265 </td><td> Supelco公司Visiprep 24-DL </td></tr><tr><td>高硼硅移液管</td><td>费舍尔</td><td> 13-678-27F </td><td> 10毫升大小</td></tr><tr><td>高硼硅巴斯德移液管</td><td>费舍尔</td><td> 13-678-8B </td><td></td></tr><tr><td>高硼硅培养管</td><td>费舍尔</td><td> 14-961-27 </td><td></td></tr><tr><td>螺纹高硼硅管</td><td> VWR </td><td> 14235-460</td><td> 8毫升容量</td></tr><tr><td>酚醛上限文化管</td><td>费舍尔</td><td> 14-823-211 </td><td></td></tr><tr><td> GC / MS自动进样器样品瓶</td><td>安捷伦</td><td> 5188-6592 </td><td></td></tr><tr><td>自动进样器样品瓶上限</td><td>安捷伦</td><td> 5185-5861 </td><td></td></tr><tr><td> GC / MS分析</td><td>安捷伦</td><td> 6890 GC / MSD 5973 </td><td>配备一个Supelcowax-10柱</td></tr><tr><td></td><td></td><td></td><td> 线虫生长培养基（NGM）：25 96以及RNA干扰板，准备1 L NGM： <table border="1"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td>去离子水</td><td> 1 L </td></tr><tr><td>氯化钠</td><td> 3克</td></tr><tr><td>琼脂糖</td><td> 17克</td></tr><tr><td>细菌用胰蛋白胨</td><td> 2.5克</td></tr><tr><td colspan="2">高压釜液循环121°C为40分钟，搅拌棒。允许冷却至60℃，搅拌下在60℃的热板上集然后添加以下内容： </td></tr><tr><td> 1 M 硫酸镁 </td><td> 1毫升</td></tr><tr><td> 5毫克/毫升的胆固醇</td><td> 1毫升</td></tr><tr><td> 1 M KH 2 PO 4（pH6.0）中</td><td> 25毫升</td></tr><tr><td> 1 M 氯化钙 </td><td> 1毫升</td></tr><tr><td> 200毫克/毫升的羧苄青霉素</td><td> 1毫升</td></tr><tr><td> 1 M IPTG </td><td> 5毫升</td></tr><tr><td colspan="2">分装到96孔的RNAi板。储存在4℃直至使用，长达2个星期。 </td></tr></tbody></table> 矩形氨苄青霉素/四环素的LB琼脂平板上（A / T）：20 A / T板，准备1 L的媒体： <table border="1"><tbody><tr><td&gt; 试剂 </td><td> 量 </td></tr><tr><td>去离子水</td><td> 1 L </td></tr><tr><td> LB培养基（美国生物） </td><td> 32克</td></tr><tr><td> 2 M的NaOH </td><td> 1.5毫升</td></tr><tr><td>细菌培养用琼脂</td><td> 2.5克</td></tr><tr><td colspan="2">高压釜液循环121°C为40分钟，搅拌棒。允许冷却至60℃，搅拌下在60℃的热板上集然后添加以下内容： </td></tr><tr><td>四环素</td><td> 3毫升</td></tr><tr><td> 100毫克/毫升氨苄青霉素</td><td> 0.5毫升</td></tr><tr><td colspan="2">分液45毫升到每一个板。在黑暗中，在4°C在使用前，长达4周的商店。 </td></tr></tbody></table> LB细菌的生长介质：4 L，足够的生长和再悬浮25 96 混合62克LB粉（美国生物ALS），4升蒸馏水，和4.8毫升2 M NaOH溶液。在1升或0.5升的等份，液体循环，40分钟高压灭菌。可以存储到1年，在室温下在密闭的瓶子。如果阴天丢弃。 100毫克/毫升氨苄青霉素 混合5μg氨苄青霉素粉末和去离子水至总体积50毫升。过滤灭菌0.2μ，免除在1毫升的等分试样，并冷冻在-20℃下直至使用。 5毫克/毫升四环素 1.25μg四环素粉末加入到250毫升的100％乙醇。搅拌均匀至完全溶解在一个不透光的容器和储存在-20°C。 200毫克/毫升羧苄青霉素 混合10克羧苄青霉素粉末和去离子水至总体积为50ml。过滤灭菌0.2μ，免除在1毫升的等分试样，并冷冻在-20℃下直至使用。 5毫克/毫升胆固醇添加1.25克胆固醇100％至250毫升乙醇。充分混合并储存在4℃至6个月。 1 M 硫酸镁 溶解在800毫升去离子水硫酸镁 120.37克。使最终体积为1高压灭菌40分钟，液体循环和储存在室温下。 1 M 氯化钙 800毫升去离子水溶解110.98克氯化钙 。使最终体积为1高压灭菌40分钟，液体循环和储存在室温下。 1 M KH 2 PO 4，pH为6.0 136.09克KH 2 PO 4溶解在800毫升的去离子水。 pH值至6.0，使终体积为1高压灭菌40分钟的液体循环并储存在室温下。 M9缓冲液：1 L-我们ROUtinely 8-10大号批 3克KH 2 PO 4，6克Na 2 HPO 4，5克NaCl，1毫升1M的用MgSO 4溶解在900毫升。将最终体积为1升100-500毫升的瓶子分装在高压灭菌40分钟，在室温下的液体循环和存储。 10 N的NaOH 400克NaOH溶解在800毫升的去离子水，可进行冷却，使最终体积为1 L存储在50ml试管中，在室温下。 1 M IPTG 23.81克IPTG加入去离子水中，将总体积为100mL。过滤灭菌0.2μ，免除在1毫升的等分试样，并冷冻在-20℃下直至使用。 鸡蛋准备漂白水/ NaOH溶液20毫升足以让一个鸡蛋准备 添加4毫升漂白剂，1毫升10 1N的NaOH，5毫升H 2 O，和10毫升M9的缓冲区50毫升聚丙烯锥形管。使用前请立即。 的尼罗红股解决方案 加入50毫升100％丙酮，25毫克尼罗河红色粉末。搅拌均匀，透光的parafilmed容器储存在室温下。可以以这种方式存储几个月。 尼罗红染色溶液：500毫升，足够用于染色30×96 -孔板 加入3ml尼罗红储备溶液至500毫升40％体积/体积的异丙醇。储存在箔包裹的玻璃瓶在室温下。使用前准备。 磷脂标准 称取7.6毫克磷脂标准，1,2 - diheptadecanoyl-sn-甘油-3 -磷酸- （1&#39;-rac-甘油），在玻璃烧杯中。溶解在20毫升2:1的氯仿：甲醇（25毫微摩尔每50μl）。查看覆盖与铝箔，直到使用。标准也可保持在-80℃，在一个密封的玻璃小瓶中，用酚醛帽1年。 甘油三酯标准 成的玻璃烧杯中，称取6.8毫克甘油三酯的标准，tritridecanoin，。溶解在30毫升2:1的氯仿：甲醇（16.7毫微摩尔每50μl）。查看覆盖与铝箔，直到使用。标准也可保持在-80℃，在一个密封的玻璃小瓶中，用酚醛帽1年。 </td></tr></tbody></table>

biochemical and high throughput microscopic assessment of fat mass in caenorhabditis elegans

线虫C.线虫已成为保守的遗传途径，调节脂肪代谢，因为它涉及到人类肥胖及其相关的病理研究的重要模式。一些以前的方法的可视化的 C开发线虫富含甘油三酯的脂肪储存，已经被证明是错误的，突出的细胞区室以外的脂滴。其他方法需要专门的设备，耗费时间，或产生不一致的结果。我们介绍一个快速，重现性好，固色剂为基础的尼罗红染色法的准确和快速检测的中性脂肪滴在C.线虫 。快速固定在40％异丙醇中的步骤，使动物完全可渗透的尼罗红，然后将其用于染色动物。此亲脂性染料的光谱性能允许它强烈和选择性荧光的黄绿色的频谱只在富含脂质的环境下，而不是在多个时极地环境。因此，可以可视化的脂滴在荧光显微镜简单的绿色荧光蛋白的成像能力，配备了后只有一个简短的尼罗红染色步骤在异丙醇。本协议的速度，可负担性和可重复性，使得它非常适合于高通量筛选。我们也表现出成对的生化测定甘油三酯和磷脂用气相色谱法，质谱分析方法。这种更严格的协议，应使用从尼罗红微观血脂测定结果的确认。我们预计，这些技术将成为新的标准，在该领域的C.线虫代谢的研究。

已采取通过脂肪染色的工作流程的成像板的一个例子是在图1中所示。具体控制高脂肪（DAF-2胰岛素受体​​RNA干扰），低脂肪（LPD-3蛋白在肠道中的RNA干扰脂质颗粒），小和低脂肪（脂肪酸合成酶FASN-1 RNAi）技术，和空载体（控制）显示的变化的荧光强度，根据动物的脂肪含量，以及相应的脂质由GC / MS分析（ 图2）确定的金额。请注意，基因失活导致发育被捕的，小动物往往会出现比成人的控制动物脂肪要低得多，不论任何脂肪代谢的连接（ 图3）。进一步的分析，包括染色分析和脂质生物化学与SPE-GCMS的野生型对照动物相似的发展阶段，必须采取措施来确保其正确性小或发育被捕了积极的命中。在FASN-1 RNA干扰（ 图2），无论是在L2或L3幼虫期的动物逮捕，并有脂肪染色低于成年对照RNA干扰的，但类似的丰富L2-L3级控制的RNAi的动物（脂肪染色％的情况下，成人的控制RNA干扰：49.9±FASN-1 RNA干扰和20.2±4.5％，2.1％L2控制RNA干扰，64.7±1.3 L3控制RNA干扰）。另外，生化分析表明，较低的TAG / PL比比L2的阶段相匹配N2控制动物（TAG / PL：22.5±2.9 FASN-1 RNA干扰和46.1±3.4阶段相匹配的对照RNA干扰，P≤0.05）。因此，在此实例中，它可以通过生化分析证实，FASN-1具有在脂质存储作用，而不仅仅是一个阶段特异性的脂肪量减少。 后续的成像蠕虫的分析在很大程度上依赖于一个计算平台，如细胞探查WormToolbox 30。对于更小的数量的图像，如ImageJ的，可自由查看从NIH，一个公开可用的图像分析平台可以被使用。在我们的分析，独特的Matlab脚本首先用于识别以及随后排除井空或有气泡，从蠕虫和区分小碎片。手动的质量控制是必需的标记的图像为上述提到的原因中排除。我们的经验发现，90百分位之间的蠕虫像素强度良好，规范化蠕虫以及在一个区域，提供了一致的度量染色强度的蠕虫病毒发展的各个阶段（ 图3）。总脂肪量集成在该区域的蠕虫病毒，相反，往往会大昏暗的蠕虫等效得分明亮的小蠕虫。因为我们的方法并不总荧光信号集成在该区域的蠕虫病毒，本身的强度百分虫大小的变化不吨偏见测得的强度。相反，在像素中的强度分布的差异的量。但是，尽管这样，明显的差异被视为在染色的不同发育阶段的动物之间的像素的强度分布，因此，重要的是要研究可比发育阶段（ 图3）对动物的任何基因的RNAi的效果。高再现性的脂肪染色法表示在图4中示出重复间的平均变异。请注意，该方法的强度是高度依赖于获得高质量的图像的每个板，均匀的照明条件下，使用相同的曝光时间，并以最小的气泡，碎片，或交叉成其他井的蠕虫。 SPE上进行预混合，纯化的标准，以确定其中的TAG的程度是可以分离从PL的（ 图5A）。在这种分析中，我们实现了100％Separation的TAG和PL，十七点〇 〇分来自从PL的脂肪酸中的TAG样本和一个相应的13时〇 〇分来自TAG在PL样品（ 图5A）的脂肪酸完全没有指明是完全没有。因此，SPE是高效分离脂质类。这种分析并不表示不同链长的所有的TAG将等效纯化由SPE和检测通过GCMS相似的效率。它仅仅保证TAG和PL血脂总彼此是完全分开的。 N2野生型动物，通过固相萃取和FAME准备一个样品GC-MS跟踪， 如图5B所示。对于这两个文件TAG和PL，峰可以识别根据保留时间和质谱父和女儿离子的上的，并根据确定的归一化的区域的各自的内部标准的峰的总面积。要确定的归一化量的TAG，区域根据所有的TAG色谱峰，除13时零零分标准，加入在一起，并除以十三时00的峰值下的面积。同样地，以确定的归一化总的PL量，所有峰下的面积，除17时00分的标准，一起加入并根据17点〇〇分峰的面积除以。的TAG和PL样品然后用归一化的值来计算最终的文件TAG / PL比样品： <img src="/files/ftp_upload/50180/50180eq1.jpg" alt="式（1）" fo:content-width="2.6in" fo:src="/files/ftp_upload/50180/50180eq1highres.jpg"/> 应当指出，数据从SPE-GCMS允许一个更复杂的分析本比简单的计算总TAG向PL的比例在每个脂质组分的脂肪酸。例如，研究者可以集成一个单峰，对应于一个单一的脂肪酸和样本之间的比较其归一化的丰度（ 例如c的daf-2的RNAi 13点○○分标准峰，归一化至总的PL的质量除以由PL 17:0标峰面积）除以18时〇 〇峰的积分面积在N2的野生型与omparing： <img src="/files/ftp_upload/50180/50180eq2.jpg" alt="式（1）" fo:content-width="4.873in" fo:src="/files/ftp_upload/50180/50180eq2highres.jpg"/> 如果研究者选择，它也将是可能的，以确定的百分比中的任意给定的脂肪酸作为脂肪酸的总量的百分比的TAG或PL馏分定量（作为一个例子十八时〇〇分在N2蠕虫在TAG馏分）： <img src="/files/ftp_upload/50180/50180eq3.jpg" alt="式（1）" fo:content-width="4.123in" fo:src="/files/ftp_upload/50180/50180eq3highres.jpg"/><img src="/files/ftp_upload/50180/50180fig1.jpg"ALT =“图1”佛：内容宽度=“3.25in”的FO：src =“/ files/ftp_upload/50180/50180fig1highres.jpg”/&gt; 图1。典型工作流程的整体实验第 1天，冷冻的RNAi库板印到安培/春节LB琼脂平板上，在37℃孵育第2天，AMP / TET板用于种子液培养的RNAi克隆深井块。第3天，细菌通过离心浓缩，并转移到96孔的RNAi板。第4天，L1幼体C.线虫被添加到在液体中的RNAi的板，并使其干燥。第7天，动物收集在液体中，用尼罗红染色，在40％异丙醇中，安装在96孔的聚四氟乙烯的幻灯片，并具有高吞吐量的显微镜成像。 <img src="/files/ftp_upload/50180/50180fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50180/50180fig2highres.jpg"/> 图2。固色剂为基础的尼罗红渍主要 <em&gt; C. N2蠕虫喂线虫的脂肪储存。代表光明的领域和GFP荧光图像显示FASN-1（小型，低脂肪），LPD-3（低脂肪），对照组（空载体），或DAF-2（高脂肪） RNAi技术克隆。样品处理按照协议（在图1中示意性）。动物固定的，在尼罗红染色，生化血脂测定和比较血脂水平所获得的成像结果。异丙醇固定尼罗红荧光水平，至少6个生物学重复获取，在第一行中所示。定量甘油三酯水平，反映整体中性脂肪存储标准化的整体磷脂水平（TAG / PL），从4生物复制，显示在第二行。重要的是要注意，从固定液尼罗红染色和生化脂质测定实现的绝对定量往往不完全匹配。在此实例中，虽然定性FASN-1（RNAi）的相似，有更多的不同的甘油三酯质量通过生物 ​​化学的方法比在显微镜下观察表明与对照组比较，则lpd-3同等不同， 的daf-2（RNAi）是少不同，虽然所有的差异是高度统计学显着测量结果重现性好。显示的数据以均数±SEM（*，P≤0.01，** P≤0.0001相对控制，由未成，双尾T-测试等方差假设）。 <img src="/files/ftp_upload/50180/50180fig3.jpg" alt="图3"/> 图3。脂肪的质量确定的固定在不同的幼虫阶段的尼罗红染色。动物上生长OP50细菌，并收集在L1，L2，L3，L4和妊娠成年发育阶段。固定染色后，图像被抓获，并使用自定义的的MATLAB脚本来确定的第 90百分位Ø分析f以上确定的蠕虫面积的像素强度。请注意的动物，血脂水平大大提高作为动物发展从L2到L4阶段，然后稍微下降的动物开始转移热量向成年阶段的种系中的增殖。显示的数据以均数±SEM重复6-8（**，P≤0.0001相对于妊娠成人，由未成，双尾T检验）。 <img src="/files/ftp_upload/50180/50180fig4.jpg" alt="图4"/> 图4。高重复性在独立的生物复制。显示的是整个生物的尼罗红的荧光强度的散点图复制组（n = 6-8）。对于每个重复中，所有的值被归一化到空载体对照的平均强度。显示的数据是平均±SEM（**，P≤0.0001相对控制，由未成，双尾T检验假设等方差）。 <img src="/files/ftp_upload/50180/50180fig5.jpg" alt="图5" fo:content-width="6in" fo:src="/files/ftp_upload/50180/50180fig5highres.jpg"/> 图5。 GC-MS色谱图，固相萃取分离的标准和野生型文件TAG和PL丰度 （A）的GC-MS分析的痕迹的SPE分离的TAG和PL标准所示。注意完全没有在PL跟踪的13点〇 〇脂肪酸的TAG跟踪的的十七点○○脂肪酸在相应的情况下，表示完全分离从PL的TAG（tritridecanoin）（1,2 - diheptadecanoyl-sn-甘油-3-磷酸-（1&#39;-rac-甘油））。 （b）代表全方位的TAG和PL从样品的N2野生型动物喂食矢量控制HT115细菌L4440空载体的RNA干扰（RNA干扰）。根据该协议，1微升的样品被注入的Agilent 6890/5973N GCMS和个别高峰被确定了支撑架很多功夫的时间和他们各自的质谱。缩写：环，环丙烷脂肪酸，ISO感光度：ISO-甲基支链脂肪酸，亚麻酸，γ-亚麻酸，ALA，α-亚麻酸; DGLA，二高γ-亚麻酸，EPA，二十碳五烯酸。 <a href="https://www.jove.com/files/ftp_upload/50180/50180fig5large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans at JoVE.com

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans | Biology | Video

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

我们提出了强大的生化和微观的研究方法<em&gt;秀丽隐杆线虫</em&gt;脂质店。一种快速，简单，固定，染色过程利用荧光脂滴成像光谱特性的亲脂性染料尼罗红。然后，我们提出的甘油三酯和磷脂固相萃取和气相色谱 - 质谱法的生化测定。

生化和高通量的微观评估的脂肪含量<em&gt;秀丽隐杆线虫</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

biochemical-high-throughput-microscopic-assessment-fat-mass

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Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

Unit 1

Chemistry of Life

Biochemistry of the Cell

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20.4K 次观看.  Massachusetts General Hospital and Harvard Medical School. 我们提出了强大的生化和微观的研究方法秀丽隐杆线虫脂质店。一种快速，简单，固定，染色过程利用荧光脂滴成像光谱特性的亲脂性染料尼罗红。然后，我们提出的甘油三酯和磷脂固相萃取和气相色谱 - 质谱法的生化测定。

视频: Biochemical and High Throughput Microscopic Assessment of Fat Mass 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

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

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, ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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 ...

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 ...