我们感谢Rayhan阿里夫和Hassaan Shahawy相机工作，Tripti Gupta和Frederike Alwes寻求协助炼细胞消融技术，Extavour实验室成员上的数据，视频和手稿的反馈。这项工作是部分由哈佛干细胞研究所的支持（种子格兰特编号SG-0057-10-00）埃利森医学基金会（新学者奖数量AG-NS-07010-10），以专家咨询小组，并以哈佛学院研究计划奖ARN。

评论说，可能是有用的在执行某些措施，表明斜体字 。 1。第1天：材料的制备<ol><li>准备以下材料（见表原材料及设备的）： <ul><li> 15厘米22厘米巴斯德移液管</li><li>金刚石划片</li><li>过滤人工海水含有1毫克/毫升两性霉素B（1:100的100毫克/毫升储液），100单位/ ml青霉素和100毫克/毫升链霉素（原液1时50分（0.0018-0.0020之间盐度）含青霉素5000单位/ ml和链霉素5000毫克/毫升） </li><li>过滤的人工海水（0.0018-0.0020之间盐度）无添加剂</li><li>住房夫妇一个大的塑料容器（至少15 x 15厘米×5厘米） </li><li>内衬的Sylgard小（3厘米5厘米）的培养皿</li><li>小（3厘米5厘米）的培养皿</li><li>一块表玻璃或玻璃多孔板</li><li>镊子（我们用钝钳来回而得米老杜蒙＃5镊子遭到意外在其他的实验室程序，我们使用镊子磨刀石，以确保镊子的末端平滑，这两个齿的长度相同，而且他们不再有已改变用途的损坏尖点。非常细镊子，如新杜蒙＃5镊子，都是不可取的，因为它们可能会损伤胚胎及/或女性）。 </li><li>巴斯德吸管的末端变宽（见下面步骤1.2） </li><li>胚胎检索工具由薄，弯曲钨丝嵌入到巴斯德吸管的薄端的（可能是由另一种替代实现，见下面1.3） </li><li>口吸管小口（见下面的步骤1.4） </li><li>玻璃针从拉玻璃毛细管制成（见下面的步骤1.5） </li></ul>没有材料对人体无毒用于本协议。然而，研究人员应该确保他们戴上手套或其他防护服装所要求的风险管理加德尔他们的机构INES。 </li><li>为了使扩大巴斯德吸管，率先得分大约15厘米的巴斯德吸管在那里的吸管开始缩小与金刚石划片，包吸管的得分区域中的Kimwipe或纸巾来保护手指的点，然后小心地分手尖的分数线。按住断头吸液管在本生灯火焰几秒钟平滑的边缘的。开口应比移液管的最大宽度稍窄。 </li><li>使钨丝解剖工具，插入一个钨丝成15cm的玻璃巴斯德移液管的端部，并保持短暂过本生灯的火焰来熔化玻璃，固定电线。使用镊子轻轻地弯曲导线的一端插入钩形状。参见图3为合适的形状，此工具的例子。 </li><li>为了使一个小口的口吸管，拉22厘米巴斯德吸管较细的一端分成两部分OV呃火焰，和折断的小端尖。插入口移液管固定器的端部。 </li><li>使玻璃针，将用于使用针拔出器的消融过程期间刺穿感兴趣的卵裂球。适当形状的针，如图4所示。使用带参数的程序2X这针被拉到一个萨特P97针拉马（热= 566，拉= 100，速度= 20，时间= 250）+ 1X（热= 566，拉= 100，速度= 100，时间= 250）。用来使针的具体程序和仪器可以不同，但​​在针必须有一个细点，但也足够坚固不打破时，对胚胎的绒毛膜推。 </li></ol> 2。第1天：收集Parhyale交配伴侣<ol><li>使用加宽巴斯德吸管（步骤1.2以上），收集交配P。 hawaiensis夫妇从大文化的容器中，并将它们转移到一个单独的包含容器C瘦人工海水。这是没有必要的，这海水进行过滤。最好是收集在后来下午交配对和离开游在室温下（20-23℃）过夜，以使第二天早晨，大多数胚胎将最近已在早期卵裂受精卵和沉积，因而级适于消融。收集至少20配合对，以确保一些女性将通过下面的早晨携早期卵裂期胚胎。 </li></ol> 3。第2天：收集Parhyale胚胎<ul><li>第二天早上，过滤注入人工海水（FASW）与CO 2。在夜间有些女性会已游过免费从他们的男性;收集这些分离的女性和麻醉他们FASW / CO 2。 剩余的未配对的男性可以从配合对容器中取出并返回到大培养。剩下的交配对可以保存，以便日后胚胎收集在当天或隔天。 </li><li>传输单个麻醉的女性携带胚胎表面皿中FASW / CO 2。所有分离的女性可能会交存鸡蛋，将通过女性的臀部透明板（ 图1A）是可见的眼睛为淡粉色或紫色球体。如果要判断胚胎在​​早期卵裂阶段，因而适合于卵裂球消融，研究者必须从育雏袋中取出胚胎，并检查他们在显微镜下。 </li><li>在解剖显微镜下观察的方法，把握臀部板沿着主体与钳的一侧。胚胎将可见在这些臀部板（ 图1B）之间的腹侧育儿袋。 </li><li>插入薄，弯曲线工具（ 图3，上述步骤1.3）进入育儿袋的后端，并轻轻抬起线向育儿袋的前端分开育雏袋覆盖物（这些修饰腹部附肢称为oostegites 30），打开内袋和松动的胚胎。胚胎是在第一个小时被解雇是在一个非常薄的，透明膜，很容易被电线工具被打乱所有封闭的左右内;产蛋后该膜消失，约2-3小时，而胚胎是不是绑定彼此或以任何方式阴从这时开始在整个胚胎发生。如果研究人员感到不便或难以操纵弯曲线工具从育雏袋中取出胚胎，另一种实现都可以使用，只要运动是温和的，没有苛刻的压力作用于胚胎或育儿袋。适合于从育儿袋取出胚胎的其他工具包括钝钳或密封和平滑末端的玻璃毛细管。 </li><li>使用一个不变的15厘米巴斯德吸管通过打开的育儿袋冲洗水，推出吨他胚胎，这应该解决的表玻璃的底部。转让女性清洁FASW（不含CO 2），以允许恢复重新引入她的主要培养前。多女性可以放置到同一个恢复盘，但允许女性以它们返回到主​​培养前完全恢复，因为它们可能会被其他动物吃掉，如果他们仍部分麻醉。 </li><li>使用一个不变的15厘米巴斯德吸管的胚胎转移到培养皿干净FASW，并查看在显微镜下确定其萌芽阶段。单独的胚胎是在第三裂解（第4阶段5），其适合于该过程。它也应该是可能的这个协议适用于任何卵裂阶段之前胚芽光盘的形成（阶段5月 7日至8日）。 </li></ul> 4。第2天：烧蚀单细胞<ol><li>填写的Sylgard林立的培养皿FASW的浅层。使用一个不变的15厘米巴斯德圆周率pette到单个胚胎转移到板。 </li><li>使用钝钳，轻轻摇动胚胎来识别小区被烧蚀（ 图2A和2B）。生殖系前体克可确定为最小micromere，它位于上最小macromere MAV的顶部。此外，G不直接接触的细胞连接 ，这是micromere直接穿过它，如中胚层前体micromeres ML和MR份额在胚胎中间的单元格边框。 先生是卵裂球克的权利， 毫升是micromere至克的左侧。 先生 ， 毫升，和连接的妹妹macromeres是外胚层前体呃 ​​， 萨尔瓦多和EP分别。 </li><li>随着来氟米特钳吨手，如果研究者是惯用右手（或右手，如果研究者是左撇子），定位与利益的权利细胞胚胎（或向左，如果研究者是左撇子），和轻轻抓住镊子之间的胚胎以使其稳定。 </li><li>使用拉玻璃针（上述步骤1.5， 图4），轻轻穿刺感兴趣的细胞。切勿将针头太远，以免针头推入相邻的单元格或感兴趣的单元格下面。针只具有穿刺绒毛膜和底层的细胞膜，并且不需要深深伸入到被烧蚀的细胞。 </li><li>交换用于刺穿胚胎为一个移液管嘴用细玻璃端部的针被拉巴斯德移液管尖（上面的步骤1.4）。握住吸管接近感兴趣的细胞产生的孔的顶端，并应用非常温和的吸力。 </li><li>很轻轻挤压胚胎的镊子。作为blasto的含量仅仅是被压出的绒毛膜通过在步骤4.4中创建的孔，它吸进移液管嘴，以允许胚胎的清晰视图。如果该孔足够大，被穿刺的细胞的细胞核可以看出出现为好。这是一个很好的检查，以确保整个单元格内容已被删除，无法再生。 </li><li>仔细观察胚胎为感兴趣的卵裂球被删除。穿刺单元的边界会退去朝着绒毛膜孔，使得可见光的底层细胞的交点。继续施加压力，直到所有感兴趣的卵裂球已被删除。使用镊子从一侧滚动胚胎到另一边，在视觉上确认所有感兴趣的卵裂球已经一去不复返了。 </li><li>用一个不变的15厘米巴斯德吸管，转移胚胎清洁FASW +（FASW + 1毫克/毫升两性霉素B，100单位/ ml青霉素和100毫克/毫升链霉素）。 </li><li>在提高单井卵裂球烧蚀胚胎一个干净的48孔板中FASW +，改变抗生素辅以FASW +日常的50％。在我们的手中胚胎生存和发展的很好，即使下面的消融，从18-28ºC的温度范围内研究者应该提高他们的胚胎为他们有一个干净的（但不一定无菌）表面上提高以最小的干扰胚胎的温度。为了能够比较卵裂球烧蚀胚胎与对照组发育事件的时间，我们建议读者参考，可用于在18ºC 4，25℃4提高胚胎的详细发育分期的信息，26ºC 5,6。 </li></ol>

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASGARD:+an+open-access+database+of+annotated+transcriptomes+for+emerging+model+arthropod+species.">ASGARD: an open-access database of annotated transcriptomes for emerging model arthropod species.</a> Database. , (2012).</li><li>Nestorov, P., Battke, F., Levesque, M. P., Gerberding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Maternal+Transcriptome+of+the+Crustacean+Parhyale+hawaiensis+Is+Inherited+Asymmetrically+to+Invariant+Cell+Lineages+of+the+Ectoderm+and+Mesoderm.">The Maternal Transcriptome of the Crustacean Parhyale hawaiensis Is Inherited Asymmetrically to Invariant Cell Lineages of the Ectoderm and Mesoderm.</a> PLoS ONE. 8, (2013).</li><li>Alwes, F., Hinchen, B., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+cell+lineage,+movement,+and+migration+from+germ+layer+specification+to+gastrulation+in+the+amphipod+crustacean+Parhyale+hawaiensis.">Patterns of cell lineage, movement, and migration from germ layer specification to gastrulation in the amphipod crustacean Parhyale hawaiensis.</a> Dev. Biol. 139, 110-123 (2011).</li><li>Ewen-Campen, B., Schwager, E. E., Extavour, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+machinery+of+germ+line+specification.">The molecular machinery of germ line specification.</a> Mol. Reprod. Dev. 77, 3-18 (2010).</li><li>Price, A. L., Modrell, M. S., Hannibal, R. L., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoderm+and+ectoderm+lineages+in+the+crustacean+Parhyale+hawaiensis+display+intra-germ+layer+compensation.">Mesoderm and ectoderm lineages in the crustacean Parhyale hawaiensis display intra-germ layer compensation.</a> Dev. Biol. 341, 256-266 (2010).</li><li>Fraser, S. E., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+metamorphosis+of+experimental+embryology.">The molecular metamorphosis of experimental embryology.</a> Cell. 100, 41-55 (2000).</li><li>Olsson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clash+of+traditions:+the+history+of+comparative+and+experimental+embryology+in+Sweden+as+exemplified+by+the+research+of+G&#246;sta+J&#228;gersten+and+Sven+H&#246;rstadius.">A clash of traditions: the history of comparative and experimental embryology in Sweden as exemplified by the research of G&#246;sta J&#228;gersten and Sven H&#246;rstadius.</a> Theory Biosci. 126, 117-129 (2007).</li><li>Rehm, E. J., Hannibal, R. L., Chaw, R. C., Vargas-Vila, M. A., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+Parhyale+hawaiensis+blastomeres+with+fluorescently+labeled+tracers.">Injection of Parhyale hawaiensis blastomeres with fluorescently labeled tracers.</a> Cold Spring Harbor Protoc. 2009, (2009).</li><li>Chaw, R., Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Independent+migration+of+cell+populations+in+the+early+gastrulation+of+the+amphipod+crustacean+Parhyale+hawaiensis.">Independent migration of cell populations in the early gastrulation of the amphipod crustacean Parhyale hawaiensis.</a> Dev. Biol. 371, 94-109 (2012).</li><li>Schmitz, E. H., Harrison, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crustacea+Microscopic+Anatomy+of+Invertebrates.">Crustacea Microscopic Anatomy of Invertebrates.</a> Microscopic Anatomy of Invertebrates. 9, 443-528 (1992).</li></ol>

作者什么都没有透露。

我们描述了一个协议，用于手动消融和从端足P的早期卵裂阶段的完整的物理去除单个卵裂球hawaiensis。我们证明使用该协议的通过从八细胞阶段的胚胎取出单个生殖系前体细胞克 ，并表明消融已成功通过确认没有G&#39;女儿细胞后的胚胎发育。这个协议可以被用来从胚胎早期卵裂阶段除去任何micromeres的。使用该协议，以除去macromeres在此阶段，或卵裂球在第一?...

在片脚甲壳类Parhyale hawaiensis已经出现在过去的十年是一个很有前途的模式生物与进化发育生物学研究1利用潜力巨大。间的节肢动物，大多数的模型系统是昆虫，和最广泛研究的是这些果蝇黑腹果蝇。D.果蝇是双翅目昆虫的一员，并作为衍生对于那些基部分枝昆虫2这样的陈列着许多胚胎学特征。此外，嵌套虫亚门Pancrustacea 3之内，这意味着昆虫有长期存在的“自然”组名为pancrustaceans内自己最亲近的亲戚，而这个群体是并系。这表明，除了基部分枝昆虫模型，其他甲壳类动物的研究来获得发展特征的进化史的一个更广阔的视野一已经这么好研究D. ND分子机制果蝇 。然而，很少甲壳类动物已经确立为发展试验实验室分析。的端足P. hawaiensis是高度易处理的实验室模型系统，适合于一系列的实验技术。端足目动物显示了许多独特的功能，他们的父母超目Peracarida（沙滩料斗，飞毛腿，和良好虾）内，并因此认为是相对这个群体的甲壳类中派生的。然而，相对容易通过Parhyale提供胚胎和功能基因操作使这个端足一个有价值的除了模式生物的当前库存。 作为实验室动物， 体育hawaiensis提供了许多优势。动物能忍受广泛的温度范围内和盐度，并在人工海水中1的大文化生存良好。这是很容易分辨本月与基于明确的形态差异，最突出的是大的，大呼过瘾，躯干前部的附肢，男性使用交配过程中掌握女性EEN男性和女性。对于胚胎学和发育的工作，P. hawaiensis有几个非常吸引人的功能。胚胎发生持续约10天的时间，性成熟大约六个星期在28ºC（但要注意，Parhyale生存以及在温度范围从大约20-30ºC，并且详细的发育分期信息可用于在18ºC 4提高胚胎，25ºC 4，和26ºC 5,6）。成人交配常年在实验室里，所以胚胎可在一年中的任何时间。雌虫2-20（取决于年龄的女性）受精卵到腹侧育雏囊位于所述第一多对腿部（ 图1A和1B）之间，并且可以收集这些胚胎S在发育早期没有杀害女性或损坏胚胎（ 图1C）。胚胎在人工过滤的海水中生存，通过孵化，可以固定为后续基因表达或组织学分析7，并有详细的临时表可以精确识别的进步，通过发展5。健壮的协议已被用于原位杂交8-15或免疫染色4,16,17进行基因表达分析，通过RNA干扰13,15或吗啉12，稳定的种系功能性敲除转基因18。使用转基因系统中，诱导表达14和增强子陷阱19的方法也可以被用来研究在巴斯德基因功能hawaiensis。而一个公开的基因组序列是当前不可用，含成绩单转录和卵子胚胎发育过程中产生的蜜蜂Ñ ​​从头组装和注释20，并存放于一个可搜索的数据库21，促进基因的发现。总之， 体育hawaiensis适用于多种实验和遗传方法来了解发展的高度听话的模式生物。 不同于D的早期合体分裂黑腹果蝇，P. hawaiensis胚胎裂解holoblastically以下受精（ 图2A）。谱系追踪分析表明，由第三切割，每个所述第三切割卵裂球的具体注定要产生1的三个胚层或中胚系6（ 图2B）。这些数据，连同微阵列数据22，细胞谱系分析6,23，和卵裂球分离实验4表明，发展潜力被隔离至少某些第三裂解卵裂球由CEL的不对称继承升命运的决定因素。因此，在该种系的前体（在Parhyale细胞谱系命名法6称为“ 克 ”）除去在8细胞期卵裂球消融的实验中，胚胎缺乏生殖细胞发育后期4，由于缺乏细胞所指示的表达该蛋白瓦萨，这是一个种系的标记中最后生动物24。与此相反，体细胞卵裂球消融的实验表明，P. hawaiensis胚胎也具有显著的监管能力，使得消融在八细胞阶段的中胚层或外胚层前体卵裂球的命运可以接管一些剩余的卵裂球25的后裔。如何调控细胞命运的更换可能发生，自治区细胞命运采用体细胞卵裂球的情况下，仍然不得而知。如卵裂球烧蚀实验胚胎学技术可以在understan是有用的鼎的相对自主性和细胞命运的决定非自治26,27，因此是在P的学习兴趣hawaiensis胚胎。 在这种证明调控更换外胚层和中胚层谱系的实验，卵裂球消融注射28和光毒性染料25的后续激励执行。虽然这种技术是在杀死注射卵裂球（次）有效，但并没有完全从胚胎中去除死细胞体。此外，差异已通过注射卵裂球用荧光示踪剂谱系28,29收集到胚胎的原肠胚形成期细胞谱系的数据，以及通过相同的胚胎阶段23的发展如下沉着卵裂球收集到的数据之间的变化。因此，完整的物理去除特定的卵裂球可能是消融的某些应用场合的首选方法。 我们以前出版的细胞谱系分析的结果胚胎中，单个细胞进行人工消融23。然而，从早期卵裂期胚胎取出单个卵裂球所需的细腻操作尚未充分说明。在这里，我们提出了一个协议的集合P的hawaiensis胚胎和从一个八细胞期胚胎的单个卵裂球的手册消融。该方法的目标是实现从胚胎完全除去细胞体的，允许观察细胞行为和胚胎发生和胚胎后发育过程中的剩余细胞的细胞命运的能力。我们的协议表示除去生殖系前体克 （ 图2C），但可以应用到任何细胞在8细胞阶段，或者早期卵裂阶段的卵裂球。原则上，该协议可以适用于从行吟诗人早期卵裂期胚胎取出单个细胞řholoblastically裂解海洋无脊椎动物。

<table border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">15 cm Pasteur pipette</td>
			<td style="width:164px;">Wheaton</td>
			<td style="width:123px;">53499-630</td>
			<td style="width:495px;">For transferring intact and blastomere-ablated embryos from dish to dish. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">22 cm Pasteur pipette</td>
			<td style="width:164px;">Wheaton</td>
			<td style="width:123px;">53499-632</td>
			<td style="width:495px;">For making mouth pipette tips. 
			VWR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">3 cm petri dishes</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:123px;">25382-334</td>
			<td style="width:495px;">Use some unaltered to collect and store embryos; coat some with a layer of Sylgard (see below) to create a working surface for blastomere ablations. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">48-well plates</td>
			<td style="width:164px;">Greiner Bio-One</td>
			<td style="width:123px;">82051-004</td>
			<td style="width:495px;">For culturing embryos following ablation. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Bottle top filters 0.2 micron</td>
			<td style="width:164px;">Nalge Nunc International</td>
			<td style="width:123px;">28199-296</td>
			<td style="width:495px;">For creating FASW (See below) 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Bunsen burner</td>
			<td style="width:164px;">VWR</td>
			<td style="width:123px;">89038-530</td>
			<td style="width:495px;">For creating tungsten wire tool and fine tip of mouth pipette. 
			VWR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:164px;">Diamond scribe</td>
			<td style="width:164px;">Musco Sports Lighting</td>
			<td style="width:123px;">52865-005</td>
			<td style="width:495px;">For creating wide-mouthed Pasteur pipettes for collecting couples. 
			VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:164px;">Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:123px;">11050-10</td>
			<td style="width:495px;">For holding anaesthetized females during removal of embryos from brood pouch. These do not have to be fine-tipped Dumont forceps - the tips can be at least as blunt as that in the forceps for which the catalogue number is listed here. 
			Fine Science Tools</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Fungizone-Amphotericin B</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:123px;">A2942</td>
			<td style="width:495px;">Add to FASW to a final concentration of 1mg/ml, and use this solution to culture blastomere-ablated embryos. 
			Sigma Aldrich</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Glass capillaries 4 inches long, 1/0.58 OD/ID (mm)</td>
			<td style="width:164px;">World Precision Instruments</td>
			<td style="width:123px;">1B100-4</td>
			<td style="width:495px;">need to include glass capillary and needle puller machine? 
			World Precision Instruments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Glass watchglass 300 mm diameter</td>
			<td style="width:164px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">70543-30</td>
			<td style="width:495px;">For use in removing embryos from the brood pouch of anaesthetized females. 
			Electron Microscopy Sciences</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Instant Ocean Artificial Sea Water (ASW)</td>
			<td style="width:164px;">Instant Ocean</td>
			<td style="width:123px;">IS160</td>
			<td style="width:495px;">Make ASW by dissolving salt in deionized water to a salinity of X. 
			Aquatic EcoSystems</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Instant Ocean Filtered Artificial Sea Water (FASW)</td>
			<td style="width:164px;">Instant Ocean</td>
			<td style="width:123px;">IS160</td>
			<td style="width:495px;">Use 0.2 micron filters to sterilize ASW. Make up in small amounts (&#60; 250 ml) as needed. 
			Aquatic EcoSystems</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Kimwipes</td>
			<td style="width:164px;">Kimberly-Clark</td>
			<td style="width:123px;">21905-026</td>
			<td style="width:495px;">For safely breaking off the fine end of Pasteur pipettes modified for collecting couples. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Mouth pipette adaptor</td>
			<td style="width:164px;">Drummond Labware</td>
			<td style="width:123px;">A5177-5EA</td>
			<td style="width:495px;">Insert the fine pulled pasteur pipette tip into this end, to be used for removing extruded cell contents during the ablation procedure. 
			Sigma Aldrich</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Mouth pipette tubing</td>
			<td style="width:164px;">Aldrich</td>
			<td style="width:123px;">Z280356</td>
			<td style="width:495px;">Cut this to be long enough to comfortably hang around your neck during the ablation procedure. 
			Sigma Aldrich</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Needle Puller</td>
			<td style="width:164px;">Sutter Instrument Company</td>
			<td style="width:123px;">P97</td>
			<td style="width:495px;">For making needles from glass capillaries for puncturing the blastomere to be ablated. 
			Sutter Instrument Company</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Penicillin-Streptomycin Solution</td>
			<td style="width:164px;">Mediatech</td>
			<td style="width:123px;">45000-650</td>
			<td style="width:495px;">Add to FASW to a final concentration of 100 units/ml penicillin and 100 mg/ml streptomycin, and use this solution to culture blastomere-ablated embryos. 
			VWR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Rubber bulb</td>
			<td style="width:164px;">Electron Microscopy Sciences</td>
			<td style="width:123px;">100488-418</td>
			<td style="width:495px;">For using Pasteur pipettes to transfer embryos from dish to dish. 
			VWR</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:164px;">Sylgard 184</td>
			<td style="width:164px;">K. R. Anderson, Inc.</td>
			<td style="width:123px;">NC9659604</td>
			<td style="width:495px;">Make up according to manufacturer&#39;s instructions. Pour a layer 2-5 mm thick into a 3cm petri dish to create a working surface for blastomere ablations. 
			Fisher Scientific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:164px;">Tungsten wire 0.004 inches diameter</td>
			<td style="width:164px;">A-M Systems</td>
			<td style="width:123px;">719000</td>
			<td style="width:495px;">Use this to make a tool for removing embryos from the brood pouch of anaesthetized females. 
			A-M Systems</td>
		</tr>
	</tbody>
</table>

ablation of a single cell from eight-cell embryos of the amphipod crustacean parhyale hawaiensis

的端足Parhyale hawaiensis是在世界范围内潮间带海洋栖息地发现了一个小的甲壳类动物。在过去的十年中，Parhyale已经成为一种很有前途的模式生物发展的实验室研究，提供了一个有用的外类群相比，以及节肢动物的研究模式生物果蝇 。而相比之下， 果蝇合胞分裂，Parhyale的早期分裂是holoblastic。使用注入早期卵裂球示踪染料命运映射表明，所有三种胚层和中胚系由8细胞阶段建立。在此阶段，3卵裂球注定要产生外胚层，三是注定要产生的中胚层和剩余的两个卵裂球为内胚层和胚系的前体分别。然而，卵裂球烧蚀实验表明，Parhyale胚胎也具有显著的监管capabilitiES，使得卵裂球烧蚀在8 - 细胞期的命运可以接管的一些剩余的卵裂球的后代。卵裂球消融以前已经描述的两种方法之一：注入和光毒性的染料或手工切除的后续激活。然而，光烧蚀杀死卵裂球，但不从胚胎去除死细胞体。因此，完整的物理去除特定的卵裂球可能是消融的某些应用场合的首选方法。在这里，我们提出了一个协议，用于从Parhyale胚胎八细胞阶段手工清除单个卵裂球，说明在保持其余卵裂球存活完好完全去除细胞体所必需的工具和手动程序。这个协议可以被应用到任何Parhyale细胞在8细胞阶段，或以其他早期卵裂阶段的卵裂球。此外，原则上这个协议可以适用于早期CLEA其他holoblastically裂解海洋无脊椎动物vage阶段的胚胎。

以下单第三裂解P的成功消融hawaiensis micromeres如本协议中所述，剩余micromeres逐渐转向它们的位置稍稍以便部分地占据以前由烧蚀卵裂球占据的空间。例如， 当 G被删除，邻近的卵裂球先生和 ML轻微移位，拿出来分享的横向单元格边框是以前在相克的直接接触（比较图2A和2C）。继成功消融，剩下的卵裂球会进入第四个卵裂...

Watch this Scientific Journal Video about Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis at JoVE.com

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

的端足Parhyale hawaiensis是一种很有前途的模式生物的甲壳类动物胚胎学和比较节肢动物的发展和演变的研究。本协议描述为从Parhyale的早期卵裂期胚胎手工清除单卵裂球的方法。

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

ablation-single-cell-from-eight-cell-embryos-amphipod-crustacean

Research

JoVE Journal

Biology

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

12.4K 次观看.  Harvard University. 的端足Parhyale hawaiensis是一种很有前途的模式生物的甲壳类动物胚胎学和比较节肢动物的发展和演变的研究。本协议描述为从Parhyale的早期卵裂期胚胎手工清除单卵裂球的方法。 

视频: Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

gentleMACS Dissociator从小鼠脾脏单细胞悬浮液的制备

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

近植入阶段人类胚胎中特罗福细胞的单细胞集合

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

尼罗罗非鱼肠单细胞悬液制备用于单细胞测序

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

植物细胞类型的分离和转录组分析

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...