图1和图2以前出版的自由基生物学及医学 （Queliconi 等。5）进行，并有由爱思唯尔举行的版权。这项工作是由支持 FUNDAÇÃO德宪法保护的单Pesquisa做斯卡德圣保罗（FAPESP），在国家研究所CIENCIAÉTECNOLOGIA DE Processos氧化还原EM Biomedicina，该核苷酸德ApoioàPesquisa去Processos氧化还原EM Biomedicina，USPHS NS064945（KN），和USPHS GM087483（KN ）。烧烤是一个FAPESP奖学金支持的博士生。

1，C.线虫生长<ol><li>准备35毫米线虫生长培养基（NGM）接种OP50细菌的琼脂平板上，按标准种植方法1。 </li><li>放置6妊娠成人到种子NGM板同步线虫 。取出后，大人〜100个卵已经奠定（〜3小时）。 </li><li>孵育所述板3天在20℃时，在该点的蠕虫会发展到年轻成年阶段和最适合于该实验。另一种协议来同步C.线虫在其他地方10描述。 </li></ol> 2。材料用于AS <ol><li> M9缓冲液（22 mM的KH 2 PO 4，42 mM的钠2 HPO 4，86 mM氯化钠，1mM的MgSO 4干燥），应通过与N 2平衡的氧气吹扫（氩气也可以被使用）在30分钟期间在实验前并保存在冰中。 </li><li>做一个小孔（0.1毫米宽）在1.5ml的微量离心管的盖子，其中，蠕虫将被放置在AS中。孔允许气体交换不保持盖子打开，因为这可能会导致过多的介质蒸发。 </li></ol> 3。缺氧/饥饿<ol><li>进行所有的实验至少一式三份。 </li><li>加入1 ml的RT，含氧M9的缓冲区含有上述长大年轻的成虫超过35毫米的钢板。要小心，以避免板去除细菌（M9添加到细菌不接种的边缘）。经过约1分钟的蠕虫，应在游泳的M9。 </li><li>大衣1毫升枪头，与BSA吹打和驱逐无菌的1％BSA溶液。 </li><li>小心倾含在M9缓冲蠕虫的板，从那里它被加在同一个地方取出M9，然后将悬浮液中制备的微量离心管中。让我们休息，直到冰蠕虫已经下降到管的底部（〜1-2分）。 </li><li>除去M9的可能的最大数量不脱离管干扰虫（留下约100微升），加入1毫升的氧气吹扫和冰镇的M9和将管在冰上，直到蠕虫沉淀到底部一次。重复最后一步3倍。在最后一次洗涤，除去M9直到〜100微升保留在管中。 </li><li>放置的蠕虫在脱氧的M9缺氧/缺氧室内部的管子（见下面的说明，第3.6.1节），或者，到定制的密封容器中（见下面的说明，第3.6.2节）。 <ol><li>如果商业缺氧/缺氧室是被使用，留下一些吹扫M9室内部开始步骤3.5之前，重复步骤3.5一旦蠕虫是在腔室内部。 </li><li>如果自定义室被使用，如何创建一个低成本的设备的详细说明，提出了在本协议的结束，已被描述的另一组先前11。简单地说： <ol&gt; <li>使两孔在250毫升百惠型容器盖，并添加管连接到它们。一个将被用于将气体混合物注入，而另一个将是气体出口进入水浴。 </li><li>将蠕虫进入腔室并用盖子密封所述腔室。 </li><li>气体以恒定的氮气流（100毫升/分钟）。 </li><li>放置在容器内的水浴在26℃。确保出口管以及浸入水中所以没有空气的回报，而没有泄漏的系统（由从腔室本身所产生的泡沫表观的）。 </li></ol></li></ol></li><li>孵化虫在26℃下20小时。 </li></ol> 4。模拟再灌注<ol><li> AS条件下20小时后，取出从孵化室管进入室内空气。 </li><li> C.移液器线虫用BSA包被的尖端（如在步骤3.3）安装到一个OP50接种35毫米NGM板。培养板在20℃下24小时。在此之后，蠕虫将准备进行评分。 </li></ol> 5。确定死亡对战现场蠕虫<ol><li>使用铂挑，轻轻触摸蠕虫的头顶。现场蠕虫会被感动后向后移动。如果蠕虫是无反应，比分为死。偶尔，蜗杆不会向后移动，但是可能表现出轻微的一侧到另一侧头部的运动。虽然技术上不是死了，这些蠕虫几乎可以肯定，在几小时内到期，应评价为死。如果等待时间过长的得分蠕虫，后代的内部孵化可以发生，这些“包包 - 的蠕虫”可破溃，使检测的尸体很困难。 </li><li>从板块中删除这两个活的和死虫，因为它们是计算以避免重复计数。活蠕虫可以移动到一个单独的板块进行行为分析。相对于总活虫量表示为幸存的蠕虫％（ 图1A </st荣&gt;）。 </li></ol> 6。触摸响应分析<ol><li>此协议的详细描述已经呈现在哈特，安妮三，编辑。行为（2006年7月3日），WormBook，编辑。该三线虫研究社区，WormBook，doi/10.1895/wormbook.1.87.1，http://www.wormbook.org。 <ol><li>进球的触摸响应，识别正在轻轻用睫毛挑（ 图3）摸虫的头部侧（靠近咽喉的中间部分）的蠕虫。 </li><li>如果蠕虫向后移动，拿到尽可能响应，如果没有，得分为无反应（ 图1B）。用10秒的时间间隔重复此步骤，10-15X的每个虫。探测至少10蠕虫每组中有准确的数据。相同的测定可用于评估向前下列轻触摸到后主体壁运动器官的变化。请务必不要触摸蠕虫的头部或尾部，因为这会刺激一个独特的行为。 </li><li>最后，在企业基因对照菌株[N2布里斯托尔野生型为阳性，MEC（mechanosensory）突变为负数- 例如 CB1338 MEC-3（e1338）IV]校准与触摸蠕虫的力量。过于苛刻的刺激会引起一个行为反应在MEC突变，过少会不会引起在野生型N2控制的效果。 </li></ol></li></ol> 7。神经修改<ol><li>以可视化的岗位作为神经修改，使用菌株表达绿色荧光蛋白在触摸响应MEC神经元4,5。这些神经元具有沿体壁运行，很容易拿下的形态异常漫长的过程。此外，本形态的信息可与神经元功能的行为的措施相结合，如上所述。可用于这个目的的一个菌株是TU2583，这是可从下线虫遗传学中心，包含uIs25集成来回的转基因表达绿色荧光蛋白米的MEC-18启动子，或者TU2562，其中包含一个集成的MEC-3 :: GFP融合。 </li><li>准备琼脂糖垫。 <ol><li>熔化的2％琼脂糖中的水，使1倍M9用10倍的库存，并保持溶液在70℃下</li><li>把该溶液的载玻片上（25毫米×75毫米）一滴（约20微升）已放置其他两个幻灯片，其中每一个具有一个单件的磁带在其底侧之间。 </li><li>将垂直于第一在2％琼脂糖溶液之上的最后搏命，并让它冷却室温。这应该形成一个垫，它是带的厚度。 </li><li>卸下顶部的幻灯片，并添加10微升的M9含0.1％四咪唑，将固定的蠕虫。 </li></ol></li><li>将部分（〜10）生活蠕虫到2％琼脂糖幻灯片。将盖玻片超过他们，并使用下一个目标100X荧光显微镜用适当的照明GFP可视化。 </li><li>破损神经元将呈现珍珠模式中的处理的胞质膜的字符串，由多个泪点（ 图2A）的，和/或异常，其中过程出现折断（ 图2B）。计数泪点和异常两种神经元对每个虫，总共使用了至少10蠕虫。 </li></ol> 8。实验室自制缺氧商会<ol><li>特百惠式的，密封的，密封的容器可以被改造为缺氧室。注意要选择一个容器，是尽可能小。一个小容器创建一个缺氧的环境更快，更容易适应在水浴中（ 图4）。 </li><li>使两个孔上的盖子（可以用螺丝刀或镊子加热并强制进入盖完成），并插入一个适合的管（ 图5A）的塑料或金属连接器。使用胶水，可以放置在水中，也变得硬干燥后，避免了运动该连接器，从而减少泄漏的可能性（ 图5B）。 </li><li>该容器应放入水浴使其完全浸没（ 图4）。浸泡容器中，用奶瓶重量或潜水的重量。 </li><li>一项所述的管连接将被用于引入气体混合物，而另一管将作为一个压力释放，因此它应在水中留下，以便将容器从外部环境（ 图4）封闭。 </li><li>空气组合物可以通过使用定制的气体质量流量控制器或混合了所需的气体的任何装置进行调制。气体流应加以控制，如高流动可以建立过度蒸发。例如，在一个250ml的容器中，气体为100ml /分将提供的蠕虫良好的空间和合适的助焊剂。 </li><li>管子的长度应保持在最低限度，因为它们可以是可渗透的气体，一llowing的O 2的入口。避免使用有机硅是很重要的，聚乙烯离心管，因为这些材料是可渗透的气体交换。我们宁愿聚丙烯或尼龙管。玻璃或金属管保持气体很好，但也很难一起工作。 </li></ol>

<ol>
	<li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Maine, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+gene+function+in+Caenorhabditis+elegans+using+RNA-mediated+interference.">Studying gene function in Caenorhabditis elegans using RNA-mediated interference.</a> Briefings Funct. Genom. Proteom. 7 (3), 184-194 (2008).</li><li>Vanden Hoek, ., L, T., Shao, Z., Li, C., Zak, R., Schumacker, P. T., Becker, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reperfusion+injury+on+cardiac+myocytes+after+simulated+ischemia.">Reperfusion injury on cardiac myocytes after simulated ischemia.</a> Am. J. Physiol. 270 (4), 1334-1341 (1996).</li><li>Ba Scott, ., Avidan, M. S., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+hypoxic+death+in+C.+elegans+by+the+insulin/IGF+receptor+homolog+DAF-2.">Regulation of hypoxic death in C. elegans by the insulin/IGF receptor homolog DAF-2.</a> Science. 296 (5577), 2388-2391 (2002).</li><li>Queliconi, B. B., Marazzi, T. B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bicarbonate+modulates+oxidative+and+functional+damage+in+ischemia-reperfusion.">Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion.</a> Free Rad. Biol. Med. 55, 46-53 (2013).</li><li>Wojtovich, A. P., DiStefano, P., Sherman, T., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP-sensitive+potassium+channel+activity+and+hypoxic+preconditioning+are+independent+of+an+inwardly+rectifying+potassium+channel+subunit+in+Caenorhabditis+elegans.">Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans.</a> FEBS Lett. 586 (4), 428-434 (2012).</li><li>Dasgupta, N., Patel, A. M., Scott, B. A., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxic+Preconditioning+Requires+the+Apoptosis+Protein+CED-4+in+C.+elegans.">Hypoxic Preconditioning Requires the Apoptosis Protein CED-4 in C. elegans.</a> Curr. Biol. 17 (22), 1954-1959 (2007).</li><li>Jia, B., Crowder, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+anesthetic+preconditioning+present+in+the+invertebrate+Caenorhabditis+elegans.">Volatile anesthetic preconditioning present in the invertebrate Caenorhabditis elegans.</a> Anesthesiology. 108 (3), 426-433 (2008).</li><li>Wojtovich, A. P., Sherman, T. A., Nadtochiy, S. M., Urciuoli, W. R., Brookes, P. S., Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLO-2+is+cytoprotective+and+contributes+to+mitochondrial+potassium+transport.">SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.</a> PloS One. 6 (12), e28287 (2011).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+methods:+synchronization+and+observation.">Basic Caenorhabditis elegans methods: synchronization and observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Fawcett, E. M., Horsman, J. W., Miller, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+defined+gaseous+environments+to+study+the+effects+of+hypoxia+on+C.+elegans.">Creating defined gaseous environments to study the effects of hypoxia on C. elegans.</a> J. Vis. Exp. (65), e4088 (2012).</li><li>Butler, J. A., Mishur, R. J., Bokov, A. F., Hakala, K. W., Weintraub, S. T., Rea, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+anaerobic+response+of+C.+elegans+using+GC-MS.">Profiling the anaerobic response of C. elegans using GC-MS.</a> PLoS One. 7 (9), 10-1371 (2012).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+cell+cycle-regulated+proteins+correlates+with+anoxia-induced+suspended+animation+in+Caenorhabditis+elegans.">Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans.</a> Mol. Biol. Cell. 13 (5), 1473-1483 (2002).</li><li>Jiang, H., Guo, R., Powell-Coffman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+hif-1+gene+encodes+a+bHLH-PAS+protein+that+is+required+for+adaptation+to+hypoxia.">The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia.</a> Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7916-7921 (2001).</li><li>Twumasi-Boateng, K., Wang, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+age-dependent+reversal+in+the+protective+capacities+of+JNK+signaling+shortens+Caenorhabditis+elegans+lifespan.">An age-dependent reversal in the protective capacities of JNK signaling shortens Caenorhabditis elegans lifespan.</a> Aging Cell. 11 (4), 659-667 (2012).</li><li>Wang, Y., Chen, J., Wei, G., He, H., Zhu, X., Xiao, T., Yuan, J., Dong, B., He, S. S. k. o. g. e. r. b. &. #. 2. 4. 8. ;. G., Chen, 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+Caenorhabditis+elegans+intermediate-size+transcriptome+shows+high+degree+of+stage-specific+expression.">The Caenorhabditis elegans intermediate-size transcriptome shows high degree of stage-specific expression.</a> Nucleic Acids Res. 39 (12), 5203-5214 (2011).</li><li>Perry, C. N., Huang, C., Liu, W., Magee, N., Carreira, R. S., Gottlieb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenotransplantation+of+mitochondrial+electron+transfer+enzyme,+Ndi1,+in+myocardial+reperfusion+injury.">Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury.</a> PloS One. 6 (2), e16288 (2011).</li><li>Chai, Y., Li, W., Feng, G., Yang, Y., Wang, X., Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cellular+Dynamics+During+Caenorhabditis+elegans+Postembryonic+Development.">Live Imaging of Cellular Dynamics During Caenorhabditis elegans Postembryonic Development.</a> Nature Protoc. 7 (12), 2090-2102 (2012).</li><li>Nehrke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reduction+in+intestinal+cell+pHi+due+to+loss+of+the+Caenorhabditis+elegans+Na+/H++exchanger+NHX-2+increases+life+span.">A reduction in intestinal cell pHi due to loss of the Caenorhabditis elegans Na+/H+ exchanger NHX-2 increases life span.</a> J. Biol. Chem. 278 (45), 44657-44666 (1074).</li></ol>

作者什么都没有透露。

如已经广泛应用于C.线虫对缺血再灌注损伤模型。一些关键点应该对该协议予以强调： 线虫耐损伤广泛，证明有必要对3伴随侮辱（热，饥饿和缺氧）使用这个系统来实现的死亡。缺氧本身并不能杀死虫子的时间14此窗口。此外，温度增加一个额外的应力，因此密切监测是很重要的。严格地说，饥饿不显著到死亡的程度有助于观察07 本身 ，但它似乎降低实验重复间的变异性。鉴于目前可以从一天到一天的显著变化，这是非常重要的，比较直接的平行样运行，并且在多天重复实验。一般情况下，结果被测量的三个独立的50-100虫板/实验条件，这些都是Ñ​​平均，视为一个单一的实验重复。一般地，间7和9重复出现足以达到或排除统计学意义。 在实验中使用的蠕虫的发育阶段也需要作为不同的阶段的敏感性的AS损伤显著15,16变化进行仔细的监测。使用年轻人是标准的，而幼虫阶段（L3和L4）蠕虫似乎对AS的（未发表数据）的破坏性影响更耐。 这个协议提供了两种方法来实现上述的实验中，1用实验室制造的装置（使用百惠型容器和气体输入11）和其他使用商业低氧室4,6。缺氧可以通过消耗氧气等手段来实现，如在别处13,17说明。使用替代技术来创造一个缺氧的环境可能会改变所需的AS孵化时间创建死亡的所需量。瞄准〜20％的生存是一个理想的起点，研究保护措施，而80％是同样理想的加剧AS的不利影响的干预措施。另一种重要的警告是时间点的观测分数死/活虫。如果分析时间延长超过24小时，数据可能会产生误导，因为死的蠕虫变得越来越难以辨认。这可能是由于虫尸变得相对透明随着时间的推移，也让一个事实，即受精的胚胎能够发育成子代验尸的尸体里面，扰乱他们，因为他们出现。 神经元形态的分析，可以进行修改，以看看蛋白表达模式18，核碎裂4和其他参数19代蠕虫菌株，表达适当的基因编码的标记。最后一个需要注意的是，该可视化把虫上滑盖后神经元突起应做不少于30分钟。动物保持麻醉状态下在幻灯片上更长的时间可以表现出的AS无关的损坏。根据跟踪和分析他们所需要的时间调整每张幻灯片的动物的数量。

线虫是，自推出以来，通过布伦纳1被广泛采用的多细胞真核模式生物线虫。它是一种廉价，简单，灵活的模型，它允许基因变异与表型之间的联系很容易改变2。 局部缺血的特征是缺乏的营养物质和氧供给到组织中，然后再灌注，当突发的活性氧产生3和大部分损伤的发生。 2002年，缺血/再灌注（IR）在C的模型线虫的开发4，涉及提交全虫缺氧，营养缺乏和热应激约20小时，然后在正常情况下24小时。尽管这种模式在技术上是一个缺氧饥饿（AS）的条件下，细胞死亡是通过那些保守的哺乳动物机制，包括再灌注诱导氧化剂损伤&lt;SUP&gt; 5。此外，类似于哺乳动物的红外光谱，损伤诱发的AS C中线虫可以通过缺血预处理6,7或麻醉药预处理8,9是可以避免的。 下面的协议演示了如何模仿IR在C线虫使用AS模型，如何得分，导致AS的形态和行为异常，以及如何适应协议的方式，允许使用一个定制的，易于构造腔替代较低的初始投资进行的实验。

<table border="0" cellpadding="0" cellspacing="0" width="1146">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">N2&#160; strain</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;"></td>
			<td style="width:453px;">Wild type strain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">TU2583 uIs25 (Pmec-18::GFP)</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;">TU2583</td>
			<td style="width:453px;">integrated fluorescent transgene used to label touch neurons</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CB1338 mec-3 (e1338)IV</td>
			<td style="width:269px;">CGC (http://www.cbs.umn.edu/CGC/)</td>
			<td style="width:208px;">CB1338</td>
			<td style="width:453px;">canonical mec-3 mutant that is touch insensitive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microcentrifuge Tube</td>
			<td style="width:269px;">Eppendorf</td>
			<td style="width:208px;">0030 120.086</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:216px;">Nikon Eclipse TE2000-U Microscope&#160;</td>
			<td style="width:269px;">Nikon USA</td>
			<td style="width:208px;">TE2000-U</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Low Temperature Incubator</td>
			<td style="width:269px;">Sheldon Manufacturing Inc.</td>
			<td style="width:208px;">Model 2005</td>
			<td style="width:453px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:216px;">Eyelash Pick</td>
			<td style="width:269px;"></td>
			<td style="width:208px;"></td>
			<td style="width:453px;">An eyelash pick can be prepared by attaching&#160; an eyelash onto a wooden toothpick, then attaching the toothpick in a glass Pasteur pipette (Figure 3)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Hypoxic Chamber</td>
			<td style="width:269px;">Coy</td>
			<td style="width:208px;">8307030</td>
			<td>Hypoxic Glove box equipped with paladium catalyst and CO2 controller.</td>
		</tr>
	</tbody>
</table>

an anoxia-starvation model for ischemia/reperfusion in c. elegans

在遗传模式生物线虫协议进行缺氧/饥饿模拟缺血/再灌注。蠕虫病毒是从细菌的食物分开，缺氧下放置20小时（模拟缺血），并随后转移到食品（模拟再灌注），一个正常的气氛。这个实验范式导致增加的死亡和神经元损伤，并且技术被提出来评估生物活力，改动触摸神经元过程的形态，以及触摸灵敏度，它表示神经元功能的行为输出。最后，对于使用公共厨房储存容器构造低氧孵化的方法进行说明。增加了一个质量流量控制单元的允许改变要作出在定制孵化器的气体混合物中，并循环水浴允许同时控制温度和可以很容易地识别泄漏。这种方法提供了一种低成本的替代方案以商业可用单位。

C.服从线虫到20小时于26℃下在自定义的实验室制造的孵化室中所描述的（第3.6.2节）带来显著的死亡率（ 图1A）5。泪点和断裂在幸存者的GFP标记的神经元突起的后续荧光成像证实形态异常的存在（ 图2）。幸存者也效果不佳，以轻体墙接触（ 图1B）。该模型已被用于多个小组，研究遗传易感性，药物干预和代谢可塑性如何影响AS相关结果4-6,8,9,12,13。 <img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig1highres.jpg" src="/files/ftp_upload/51231/51231fig1.jpg" /> 图1。C.线虫后生存和触摸响应AS A）蠕虫的百分比，表现出24小时后的AS生存。在N2菌株是野生型的遗传背景，并KWN85包含一个集成的转基因的标签MEC神经元GFP B）响应触摸活C的刺激线虫 （N2株）后的AS。 <a href="https://www.jove.com/files/ftp_upload/51231/51231fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig2highres.jpg" src="/files/ftp_upload/51231/51231fig2.jpg" /> 图2。后触摸神经修改。触控神经元（PLML和PLMR）异常，如曲折的过程和休息（A）或绿色荧光蛋白聚集体的过程（B）的累积生存进行了监测麻醉C。 ELEG<html> ANS后的AS。 <img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig3highres.jpg" src="/files/ftp_upload/51231/51231fig3.jpg" /> 图3。睫毛选秀权。一种组装式睫毛挑。在插图中，睫毛的细节粘在牙签的木材。 <img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51231/51231fig4highres.jpg" src="/files/ftp_upload/51231/51231fig4.jpg" /> 图4。实验室自制缺氧室水池内的一个封闭的容器准备开始实验。箭头表示气体的入口和出口，并用于保持漂浮在瓶的重量。 231fig5.jpg“/&gt; 图5。容器盖的内部和外部侧的详情。容器盖的A）外部侧，表示附加到先前作出孔B）的容器盖的外侧面，指示附加到先前作出的孔的管接头的管及其接头。

Watch this Scientific Journal Video about An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans at JoVE.com

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

的协议，描述了使用缺氧/饥饿在C线虫模型缺血/再灌注。功能结果包括死亡率上升，在GFP标记的神经元突起明显异常，需要神经功能受损的行为反应。

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

an-anoxia-starvation-model-for-ischemiareperfusion-in-c-elegans

Research

JoVE Journal

Biology

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

9.8K 次观看.  Universidade de São Paulo. 的协议，描述了使用缺氧/饥饿在C线虫模型缺血/再灌注。功能结果包括死亡率上升，在GFP标记的神经元突起明显异常，需要神经功能受损的行为反应。 

视频: An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

科研

生物学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

C. Elegans生理应激反应的测量

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...