我们承认L. Brino（IGBMC高内涵筛选设备，伊尔基希，法国）为我们提供了抗Giantin抗体，M. Labouesse实验室。为C.线虫 （IGBMC）和B塞拉芬实验室。对芽殖酵母（IGBMC），E帕卢赫和A.海曼荧光灯HeLa细胞（MPI-CBG，德累斯顿），J·莫斯利（达特茅斯医学院）和JQ吴（俄亥俄州立大学）的裂殖酵母细胞; A.饭店Hoel和F Evenou实验的帮助下，里克C.（IBMC，斯特拉斯堡，法国）的技术的帮助下，和JC JEANNOT（飞秒日，法国）在微细的帮助。这项工作是由来自法国国家科学研究中心，斯特拉斯堡大学，Conectus资金的支持，拉基金会倒拉RECHERCHE MEDICALE和斯特拉斯堡的CI-FRC。

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我们什么都没有透露。

副本成形，为了制造"eggcups"使用。的制造过程中不需要清洁室;它是容易和简单，但也有一些实践可能是需要的。具体地，释放PDMS压模是为了生产高品质&#39;eggcups&#39;的一个大的面积中最关键的步骤。由于这个原因，特别小心在这一步骤才能作出。如果这一步失败反复，考虑到优化之前的硅烷化和等离子结合等离子清洗机的参数。硅烷化不足，会导致邮票的PDMS膜粘着强。如果发生此种情况，培养时间与硅烷化试剂可以增加。需要注意的是其它技术和材料可应用于制造的"eggcups&#39;，它可以用一个大范围的配位体（纤连蛋白，明胶，胶原等 ）的官能化。特别是，在聚苯乙烯微腔可以很容易地通过硫化铜制成汤姆制热压印技术。这确保生物相容性和在标准培养皿得到的结果直接比较。同样地，特别护理和实践都需要以优化的填充比率。特别地，漂洗步骤是为了确保没有过量细胞的适当填充，在信号有助于噪声和背景的关键。如果细胞是从空腔容易地除去，可以考虑改变腔的大小或深度。 &#39;Eggcups&#39;提供3D状结构用一个简单的协议细胞和高含量筛选测定法。细胞器和未知活动进程使用标准培养测定可通过在个别的微腔（&#39;eggcups&#39;）插入的单细胞的方法很容易地可视化。取决于模型系统上，大小，形状和它们的尺寸可以很容易地适应。在这种方式哺乳动物细胞，裂殖酵母，出芽酵母和C.线虫 ç一个被操纵和研究，以及任何胚胎例如果蝇 ，小鼠或人类胚胎在体外受精 ，或干例如细胞。 在此设置中单细胞被捕获。这是相对于在体内遇到的上皮组织。然而，这种环境可以在我们的&#39;eggcups&#39;通过与钙黏着蛋白涂覆的侧壁以模仿采用更为灵活的弹性体的细胞 - 细胞接触再现。焦接触将纤连蛋白的沉积在孔的底部被提升。粘附分子的这些各分布应允许在再现体内遇到的蜂窝环境。通过这种方法的一个会接近生理条件。 在我们的测定培养基交换保证。在EC细胞由于缺乏培养基交换的执行短期和长期实验时不显示任何降解。还需要注意的是细胞的EC CAn为直至汇合培养虽然主要关心的是，当单个细胞或胚胎腔进行隔离。 细胞器或整个有机体的方向是透露出新的信息。我们发现在cytokinetic环肌动蛋白和肌球蛋白的不同动态。虽然在裂殖酵母和哺乳动物细胞的cytokinetic环由类似的关键部件，我们将展示这种设置，他们的具体动态是不同的17。这是支持的结果，在这两个系统的闭合机构是不同的。开发和研究这样一个假设，该单元的取向是不可缺少的。在将来的研究中，该装置也可以用来研究在细胞的细胞器组织相关的其他活动。 除此之外，该技术可以在发育生物学大用场。细长的胚胎可以很容易地导向，观察或以限定的方向进一步处理。可能是我们测定不会强加胚胎极性，但高填充比率将允许以提取所需的读出以可靠的方式。共有"eggcups"可能是高含量放映的好设备。 其它培养测定已经被提出。这些方法的范围从多个小区在多孔板2D尺寸，沉积在具有相同形状微图案化粘合剂图案单细胞。然而，它们都不是适合于克服对细胞的细胞器和动力过程1的观察上文详述的限制。 我们的系统今后的改进将使"eggcups"面向产业用途的适用性。作为一个例子，在制药公司药物筛选应用需要使用多孔板14,28的;实施"eggcups"到这样的平台将有可能提高的可靠性测试和结果。因此，高含量，筛选分析将通过使用机器人制药公司（和学术研究实验室）的常用automatized流程进行。这将确保可重复性和可靠性低变异性。基于三维细胞培养测定法的一些商业产品已经出现在市场上突出这种测定法的重要性。最后，这些装置打开个性化药物新观点:来自患者的细胞可以被放置在&#39;eggcups&#39;，和治疗鸡尾酒能在生理环境中进行测试;生物标记读出将使预期要给予患者29的最佳治疗。共细胞和胚胎的物理形状指导腔的结构，我们希望该设备，这种方法将被广泛流传于未来。

当前在体外基于细胞的测定是二维（2D）。这种构造是不自然的哺乳动物细胞，因此不是生理有关1;细胞显示的形状，大小和异构表型的多样性。当应用于筛选应用，如平面和细胞器的极端的表型（应力纤维，特别是）内的无序分布它们呈现额外严重的局限性。这是在药物测试，在高预算每年花在临床试验尤为重要。当因为在药物筛选的初期阶段人工2D培养条件适用于动物模型大多数这些药物​​虽然失败。另外，通过使用这种方法，具体细胞器无法正常显现，如细胞有丝分裂期间cytokinetic肌动球蛋白环，一般认为是在平面垂直演进到观察平面结构。一些新的二维测定人为了克服上述缺点和重要的见解上细胞骨架组织已经观察到2,3被提出。但是，这些检测仍然存在一个严重的局限性:细胞表现出非常价差表型相反的是， 在体内 ，其中细胞呈现出3D架构观察。与培养法相关的这些文物可能引发非生理功能，如增强的应力纤维1,4,5。 时相比2D环境6,7-三维细胞培养测定提供多个优点。它们是生理上更相关的，并且结果是因此有意义。作为一个例子，嵌在水凝胶细胞显示3D状结构，但它们的形态从一个小区的不同而不同8,9。然而，它们的形态从一个小区到另一个，其中筛选应用复杂不同。另一种策略是嵌入单细胞微制造的腔10,11。然后细胞位置，形状，极性和内部细胞组织可以成为标准化。除了 ​​提供类似3D的架构，细胞，微腔还允许高内涵筛选研究10,12-14;单电池可以订购成微阵列和细胞器和它们的演进可以并行进行观察。这个规律提供了较低的细胞数量和更好的时间/空间分辨率良好的统计数据。有用的化合物是更容易识别可靠。 在这项研究中，我们显示了高内涵筛选应用10,12,13制作和全新的3D般的单细胞培养系统中的应用。该装置由弹性微腔（10 5腔/厘米2），创造&#39;eggcups"（EC）的阵列。在这项工作中的尺寸和EC的总体积进行优​​化，个人NIH3T3细胞和HeLa细胞的典型体积在细胞分裂过程。圆柱形 - - 腔形态被选择为活动进程的可视化的正确东方细胞形状。复制品成型用于图案EC的阵列上附着在玻璃的薄聚二甲基硅氧烷（PDMS）层盖玻片15,16。细胞通过离心在欧盟出台。我们在2D使用相同的细胞相比在这里报告的观察和三维（EC）细胞器（应力纤维，高尔基体和胞核）正常化（平）表面。我们还报告，积极动力过程的观察，如cytokinetic肌动球蛋白环的细胞有丝分裂过程中17封。最后，我们表明与刚性壁，如出芽酵母，裂殖酵母和C的其它系统这种方法的结果线虫胚胎这证实了我们的方法是否适用于广泛的模型系统。 接下来我们提出一个详细而详尽的protocol为了制造和应用三维微细加工的"eggcups"。我们的方法是简单，并不需要一个干净的房间。我们预计，这一新方法将成为药物筛选试验和个性化的医学特别有意思，在更换培养皿。最后，我们的设备将用于癌症18或在基础研究19研究的细胞响应的分布对外部刺激，例如有用。

<table><tbody><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;0 (ultrapure)</td><td>Millipore</td><td>-</td><td>Use always fresh water.</td></tr><tr><td>Parafilm (plastic film)</td><td>Bemis</td><td>PM-999</td><td>Adhere Parafilm to the lab bench using some water droplets and ensure a perfect surface flatness.</td></tr><tr><td>Photo-mask</td><td>Selba</td><td>-</td><td>&lt;a href=&quot;http://www.selba.ch/&quot; name=&quot;RANGE!D4&quot;&gt;http://www.selba.ch&lt;/a&gt;</td></tr><tr><td>Silicon wafer</td><td>Siltronix</td><td>-</td><td>&lt;a href=&quot;http://www.siltronix.com/&quot;&gt;http://www.siltronix.com/&lt;/a&gt;</td></tr><tr><td>SU-8 photoresist</td><td>MicroChem</td><td>2000 series</td><td>&lt;a href=&quot;http://www.microchem.com/Prod-SU82000.htm&quot;&gt;http://www.microchem.com/Prod-SU82000.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>SU-8 developer</td><td>MicroChem</td><td>-</td><td>&lt;a href=&quot;http://microchem.com/Prod-Ancillaries.htm&quot;&gt;http://microchem.com/Prod-Ancillaries.htm&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>working in a fumehood is required; check the data sheet from the manufacturer for more information</td><td></td><td></td><td></td></tr><tr><td>2-propanol</td><td>Sigma-Aldrich</td><td>19030</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/i9030?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Sigmacote (siliconizing reagent )</td><td>Sigma-Aldrich</td><td>SL2-25ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/sl2?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>harmful, working in a fumehood is required; check the data sheet from the manufacturer for more information.&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Chlorotrimethylsilane (TMCS)</td><td>Sigma-Aldrich</td><td>386529-100ML</td><td>http://www.sigmaaldrich.com/catalog/product/aldrich/386529?lang=fr&amp;amp;region=FR&amp;nbsp;&amp;nbsp;</td></tr><tr><td colspan="4">TMCS produces acute inhalation and dermal toxicity, and is highly flammable (with ignition flashback able to occur across considerable distances), consequently it should be used in a fume cupboard away from sources of ignition</td></tr><tr><td>Nitrile gloves</td><td>Kleenguard</td><td>57372</td><td>&lt;a href=&quot;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&quot;&gt;http://www.kcprofessional.com/products/ppe/hand-gloves/thin-mil-/57372-kleenguard-g10-blue-nitrile-gloves-m&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Glass coverslips #0</td><td>Knittel glass</td><td>KN00010022593</td><td>&lt;a href=&quot;http://www.knittelglass.com/index_e.htm&quot;&gt;http://www.knittelglass.com/index_e.htm&lt;/a&gt;</td></tr><tr><td>Very fragile. Manipulate gently.</td><td></td><td></td><td></td></tr><tr><td>Sharp straight tweezers</td><td>SPI</td><td>0WSSS-XD</td><td>http://www.2spi.com/catalog/tweezers/t/elec7</td></tr><tr><td>50 ml tube</td><td>BD Falcon</td><td>352070</td><td>http://www.bdbiosciences.com/cellculture/tubes/features/index.jsp</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 kit</td><td>&lt;a href=&quot;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&quot;&gt;http://www.dowcorning.com/applications/search/default.aspx?R=131EN&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>The package contains both PDMS base and curing agent. Similar elastomers are available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>Microscope glass slides</td><td>Dutscher</td><td>100001</td><td>&lt;a href=&quot;http://www.dutscher.com/frontoffice/search&quot;&gt;http://www.dutscher.com/frontoffice/search&lt;/a&gt;</td></tr><tr><td>Available from multiple companies.</td><td></td><td></td><td></td></tr><tr><td>DMEM high-glucose medium</td><td>Fisher Scientific</td><td>41965-039</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?LBCID=12301479&amp;amp;keyWord=41965-039&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Bovine calf serum</td><td>Sigma-Aldrich</td><td>C8056-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/c8056?lang=en&amp;amp;region=CA&lt;/a&gt;</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Fisher Scientific</td><td>25200-072</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=25200-072&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS 1x</td><td>Fisher Scientific</td><td>14200-067</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=14200-067&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>PBS is at 10x and should be diluted to 1x using ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>L-15 medium</td><td>Fisher Scientific</td><td>21083-027</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=21083-027&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=PROD&lt;/a&gt;</td></tr><tr><td>Medium for atmospheres without CO&lt;sub&gt;2&lt;/sub&gt; control</td><td></td><td></td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F1141-5MG</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=F1141-5MG&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Penicillin &amp;amp; Streptomycin</td><td>Fisher Scientific</td><td>15140-122</td><td>&lt;a href=&quot;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&quot;&gt;http://www.fishersci.com/ecomm/servlet/Search?keyWord=15140-122&amp;amp;store=Scientific&amp;amp;nav=0&amp;amp;offSet=0&amp;amp;storeId=10652&amp;amp;langId=-1&amp;amp;fromSearchPage=1&amp;amp;searchType=CHEM&lt;/a&gt;</td></tr><tr><td>Petri dish P35</td><td>Greiner</td><td>627102</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/en/row/articles/catalogue/article/144_11/12885/&lt;/a&gt;</td></tr><tr><td>Petri dish P60</td><td>Greiner</td><td>628163</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/145_8_bl/24872/&lt;/a&gt;</td></tr><tr><td>Petri dish P94</td><td>Greiner</td><td>633179</td><td>&lt;a href=&quot;http://www.greinerbioone.com/&quot;&gt;http://www.greinerbioone.com/nl/belgium/articles/catalogue/article/146_8_bl/24882/&lt;/a&gt;</td></tr><tr><td>Paraformaldehyde 3 %</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sial/p6148?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>Harmful in-particular for the eyes, working in a fumehood is required; check the data sheet from the manufacturer for more information.</td><td></td><td></td><td></td></tr><tr><td>Triton 0.5 %</td><td>Sigma-Aldrich</td><td>93443-100ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=93443-100ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Phallodin-Green Fluorescent Alexa Fluor 488</td><td>InVitrogen</td><td>A12379</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&quot;&gt;http://www.lifetechnologies.com/order/catalog/product/A12379?CID=search-a12379&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>dissolve powder in 1.5 ml methanol</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 647</td><td>InVitrogen</td><td>A21245</td><td>1:200 dilution in PBS 1x</td></tr><tr><td>rabbit polyclonal anti-Giantin</td><td>Abcam</td><td>ab24586</td><td>1:500 dilution in PBS 1x</td></tr><tr><td>&lt;a href=&quot;http://www.abcam.com/giantin-antibody-ab24586.html&quot;&gt;http://www.abcam.com/giantin-antibody-ab24586.html&amp;nbsp;&lt;/a&gt;</td><td></td><td></td><td></td></tr><tr><td>rabbit anti-anillin</td><td>Courtesy of M. Glotzer, Published in Piekny, A. J. &amp;amp; Glotzer, M. Anillin is a scaffold protein that links RhoA, actin, and myosin during cytokinesis. Current biology 18, 30&amp;ndash;6 (2008).</td><td></td><td>1:500 dilution in PBS 1x</td></tr><tr><td>Anti-phosphotyrosine</td><td>Transduction Lab</td><td>610000</td><td>http://www.bdbiosciences.com/ptProduct.jsp?ccn=610000</td></tr><tr><td>Cy3 goat anti-rabbit&amp;nbsp;</td><td>Jackson Immunoresearch</td><td>111-166-047</td><td>&lt;a href=&quot;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&quot;&gt;http://www.jacksonimmuno.com/catalog/catpages/fab-rab.asp&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1:1,000 dilution in PBS 1x</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417&amp;nbsp;</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&quot;&gt;http://www.sigmaaldrich.com/catalog/product/sigma/d8417?lang=fr&amp;amp;region=FR&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>1 mg/ml for 1 min</td><td></td><td></td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G2025</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=G2025&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410-500ML</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&quot;&gt;http://www.sigmaaldrich.com/catalog/search?interface=All&amp;amp;term=M8410-500ML&amp;amp;lang=en&amp;amp;region=CA&amp;amp;focus=product&amp;amp;N=0+220003048+219853082+219853286&amp;amp;mode=match%20partialmax&lt;/a&gt;</td></tr><tr><td>HeLa cells</td><td>-</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>NIH3T3 cells</td><td>ATCC</td><td>-</td><td>Mammalian cells are available from many companies. See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>Fission yeast&amp;nbsp;</td><td>-</td><td>-</td><td>For details on strains, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; worms</td><td>-</td><td>-</td><td>For details, contact the corresponding author.&amp;nbsp; See also &lt;strong&gt;Table 1&lt;/strong&gt;</td></tr><tr><td>YES (Agar) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-732</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-732&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>YES (Media) + 5 Supplements included</td><td>MP Biomedicals</td><td>4101-522</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4101-522&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow the instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>EMM (Media)</td><td>MP Biomedicals</td><td>4110-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4110-012&amp;amp;s=Search&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>For preparation: follow&amp;nbsp; instructions as given on the box</td><td></td><td></td><td></td></tr><tr><td>Filter sterilized EMM (Media) - Only for imaging</td><td>MP Biomedicals</td><td>4110-012</td><td>For preparation: follow instructions as given on the box. Filter sterilize the media using a 0.22 &amp;micro;m filter instead of autoclaving. This gives transparency to the media and reduces the autofluorescence.</td></tr><tr><td>Supplements (for EMM)</td><td>MP Biomedicals</td><td>4104-012</td><td>&lt;a href=&quot;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&quot;&gt;http://www.mpbio.com/search.php?q=4104-012&amp;amp;s=Search&lt;/a&gt;</td></tr><tr><td>&amp;nbsp;(Add 225 mg/L into the EMM media before autoclaving or filtering)</td><td></td><td></td><td></td></tr><tr><td>Stericup and Steritop Vaccum driven sterile filters</td><td>Millipore</td><td>-</td><td>&lt;a href=&quot;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&quot;&gt;http://www.millipore.com/cellbiology/flx4/cellculture_prepare&amp;amp;tab1=2&amp;amp;tab2=1#tab2=1:tab1=2&lt;/a&gt;</td></tr></tbody></table>

ordering single cells and single embryos in 3d confinement: a new device for high content screening

生物细胞通常在平面（2D）表面观察。这种条件是不生理和表型和形状是高度可变的。根据在这种环境中的细胞筛选具有因而严重的局限性:细胞器显示极端的表型，细胞形态和尺寸是异质和/或特定的细胞器无法正确显示。此外， 在体内细胞位于3D环境;在这种情况下，细胞表现出不同的表型的，主要是因为它们与组织的周围的细胞外基质相互作用的。为了标准化和产生单个细胞中对基于细胞的测定生理相关的3D环境的次序，我们在这里报告的装置的用于体外三维细胞培养物的微加工和应用。此装置由微腔的2D阵列（通常是10 5的空腔/厘米2）中 ，填充有单细胞或胚胎。细胞POSI化，形状，极性和内部细胞组织变得那么标准化呈现出3D架构。我们使用复制品成型图案的微腔的阵列，&#39;eggcups&#39;，到薄聚二甲基硅氧烷（PDMS）粘附在盖玻片层。腔体上布满了纤维连接蛋白更容易粘附。细胞通过离心插入。填充百分比为每个系统允许高达80％的最优化。细胞和胚胎存活率证实。我们应用此方法对细胞器，如细胞核和高尔基体的可视化，并研究活动进程，如cytokinetic环的细胞有丝分裂期间的关闭。该器件允许cytokinetic闭环和压实表型的高尔基体细胞核对准期间的新功能，如周期性积累和肌球蛋白和肌动蛋白的不均匀性的鉴定。我们的特点的方法哺乳动物细胞，裂殖酵母，芽殖酵母，C.线虫 W¯¯在每种情况下i个具体的适应。最后，该装置的特性使得它特别有趣的药物筛选测定法和个性化的药物。

该&#39;eggcups&#39;（EC）是一种新型的高含量筛选方法，它允许导向细胞和胚胎的可视化在3D环境。此外，某些细胞过程，这是很难在标准2D（平面）培养物来观察，可以通过这种新的方法观察到的。 图1a显示了EC微细加工步骤的概要（参见章节1中的上述协议）。该方法简便，快捷，高效，无需特殊设备的任何要求。 图1b和1c显示了一个大型的图片和"eggcups"分别放大的扫描电子显微镜图像。如可以观察到的，它们的形状和大小是非常规则。这种方法是非常灵活的;不同形状和尺寸可以容易地制造，并且适合于不同的模型系统。以下面的方式被选择的&#39;eggcups&#39;尺寸:尺寸其中发生分裂的细胞，测定二维表面:它们具有球形形状并且它们的直径取作的良好指示对于EC直径。例如细胞分裂过程中&#39;eggcups&#39;长和定向沿其长轴细胞。这个尺寸取决于系统 - 细胞和胚胎 - 所以此尺寸应在每种情况下进行评估。 图2示出所需要的材料（ 图2a）和步骤一步协议（ 图2b）关于如何使用"eggcups&#39;（也参见在上述协议第2节）。欧共体与权益（或其他模型系统）细胞的充盈是非常简单和快速。通常情况下，它需要小于20-30分钟，其中还包括对细胞胰蛋白酶消化的时间。在填充后，样品可以用来研究活动进程（实时成像）或可以是固定的和染色intere的细胞器的可视化ST（也见第3和第4在上述的协议）。 在平坦的表面，细胞表现出异质反应和细胞器的极端表型。事实上，已经表明，肌动蛋白应力纤维（和其它细胞器）是在培养条件1工件。为了证明这一假设，我们培养既三维&#39;eggcups&#39;和上平坦表面和比较不同细胞器，即肌动蛋白应力纤维，高尔基体和核的表型NIH3T3细胞。 图3显示的细胞是如何组织的一个例子在这两种配置。在乳油，将细胞分布在示出一个均匀的球形样表型（ 图3a）的有序数组。上平坦的表面，细胞显示典型的无序，扩散和多相形态（ 图3b）。也有在骨架结构显著差异。特别是，细胞R上16; eggcups&#39;显示相比平坦表面应力纤维的数量的减少。这是在三维进一步证实重建，其中没有明显的应力纤维是可见的（参见图3c - D）图像。这证实了一些蜂窝结构在2D培养物放大。这也与在体内 ，其中应力纤维不能被识别进行观察一致。 高尔基体还示出了在根据不同的培养条件它们的表型显著变化（参见图4）。二维文化高尔基体通常显示的扩展型&#39;拥抱&#39;原子核周围，而在"eggcups"它显示了一个更紧密的表型（ 见图4A - B）。为了模拟一个药物筛选操作，我们还评估药物对细胞上两种环境中培养的效果。我们选择成为内地II型肌球蛋白y方法，因为它破坏了肌动蛋白应力纤维和可能对高尔基形态（参见图3c - D）的效果。因为高尔基毗邻细胞核，这种药物也可能会对它的结构的效果。我们首先观察到，与此药物处理的细胞相比，野生型（WT）细胞（参见图3c - D）表现出较少定期和均一的形态。然后，我们比较和定量上&#39;eggcups&#39;和在平坦的表面（参见图4c）中观察到的高尔基表型。我们观察到，在二维表面细胞多呈扩展的表型而在"eggcups"细胞表现出较为致密的表型。我们并没有观察到，虽然WT和II型肌球蛋白处理的细胞之间的显着差异。 最后，在2D表面细胞核是随机取向，而对于在EC细胞是正交相取向于XY平面中都WT和II型肌球蛋白治疗的细胞（ 见图5A - C）。这突出该装置的定向细胞器，类似于用于定向在酵母和哺乳动物细胞10,12,13的cytokinetic环的观察平面的方法的前应用的强度。我们终于研究细胞核球形如何（定义为ψ= [π 三分之一 6V N 2/3] / A N，其中V n是细胞核的体积和正其表面积）受到影响取决于培养条件和在用II型肌球蛋白的细胞的治疗; 图5d示出ψ的相应分布。我们并没有观察到的野生平 VS WT EC的差异，这表明欧盟在不影响细胞的正常球形。然而，我们观察到比较WT 乳油时差Blebb EC表明欧共体被揭示在2D掩蔽药物的实际效果。 使用"eggcups&#39;活细胞研究还允许这是不标准的文化可见新颖的活动进程的鉴定。我们铺板的细胞在EC和可视化的细胞分裂。 图6显示了cytokinetic环闭合的图像的细胞有丝分裂期间的序列。该&#39;eggcups&#39;装置允许该环的一个完整的可视化，而 ​​标准的2D培养物只示出了对应于一个单一平面10两个区域。使用2D培养Z堆叠图像的序列的环的重建可以做到27，但重要的信息丢失。质量被减少由于低Z的分辨率和动态过程不能得到解决。肌动蛋白和肌球蛋白都在力产生细胞分裂的关键蛋白。它们的动态可知道BÈ成像和在2D培养（ 图6a）的研究，而用"eggcups&#39;则立即显现出来。我们已经确定了新的结构和过程:在HeLa细胞中，我们发现肌球蛋白17的周期性积累。这些积累径向移动作为环正在关闭（ 图6b）。在裂殖酵母中我们也发现肌球蛋白和肌动蛋白的不均匀性（ 图6c，右）17。相反，我们在HeLa细胞中看到，他们关闭期间旋转环上。速度是在微米分钟-1的范围，就不会为z重建与标准显微镜解析。最后cytokinetic环可以通过染色的部件进一步被研究。我们发现，有磷酸的环中（ 图6d）的附近的积累。我们也可以显示anillin在环（ 图6E）colocalizing。通过染色的细胞在这个方向，WË表明anillin显示也是不均匀分布。 该&#39;eggcups&#39;也被应用到不同的模型系统:我们报道哺乳动物细胞，裂殖酵母，但我们也测试了芽殖酵母和C.线虫 （ 见图7A - E）。在这种情况下，该协议被改编为在培养基方面每个特定的系统，空腔大小和形态（ 见表1）。作为一个例子，锥形V形"eggcups&#39;是用于有效地12固定裂殖酵母的最佳形态，代替完全圆柱形（或U形），用于哺乳动物细胞13的形状。这使得在生命科学研究领域的潜在应用测试不同的细胞骨架药物的疗效。这证明了开发方法的灵活性和可靠性。 此外，细胞的高度有序布置允许一个方便，自动读出单个细胞的荧光。我们通过将在&#39;eggcups&#39;（ 图8a）表达GFP NIH3T3细胞说明这一点。单元位置可以容易地识别和相应的表达水平进行测量。 图8b示出荧光信号的分布。这可以应用到任何读出（免疫荧光，荧光报道在细胞例如）。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51880/51880fig1.jpg" /> 图1:"eggcups"的制作 （ 一 ）复制成型"eggcups"的制作过程示意图说明:（1）倒在SU-8模具液体的PDMS和治愈它。 （二）剪下邮票从表面小心取出，然后等离子激活它silanize它。 （三）倒在硅烷化邮票的液体硅橡胶ð离心它以获得薄的PDMS层。 （四）固化的PDMS层之后，等离子体同时激活，与PDMS覆盖印模和玻璃盖玻片。 （五）血浆地结合运用柔和，均匀的压力。 （六）等离子体结合后，小心地取出邮票揭开"eggcups"表面。 （七）为简化在接下来的步骤中的处理，增加一个小的PDMS手柄件。通过用液体PDMS胶合它和（ⅷ）固化它，然后在烤箱结合的PDMS片盖玻片。 （ 二 ）25毫米盖玻片与PDMS&#39;eggcups&#39;和手柄的图像。 PDMS"eggcups"（ 三 ）扫描电子显微镜图像。 "eggcups&#39;的中心之间的距离为30微米，其直径约25微米。（左）顶视图（右）"Eggcups&#39;被切割成图像内的一部分。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig1large.jpg" target="_blank">请点击此处争夺</a><html> WA更大的版本这个数字。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51880/51880fig2.jpg" /> 图2:（ 一 ）所需EC填充单元。 （1）50毫升管; （2）的圆柱形片（俯视图和侧视图）; （3）细胞培养基; （4）&#39;eggcups&#39;; （5）尖锐的镊子。 （ 二 ）欧盟灌装过程示意图。 （ⅰ）一个圆筒形件首先被引入到50ml管中并填充有13个ml的细胞培养液中。接着，（ii）该&#39;eggcups&#39;被轻轻敷使用尖锐的镊子使用小的PDMS片操纵EC圆筒件的顶部。 （三）在适当的密度细胞吸取到欧盟的顶部。 （ⅳ）将细胞在离心&#39;eggcups&#39;引入的。 （五）最后，将样品轻轻地从管释放出来，这是准备使用。<html> M /文件/ ftp_upload / 51880 / 51880fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51880/51880fig3.jpg" /> 图3:三维&#39;eggcups&#39;和2D平坦表面细胞表型的比较。共聚焦显微镜（25X水客观，0.95的NA，徕卡）是（a）EC NIH3T3细胞形成的有序阵列，并示出一个均匀的球形表型，并在（B）标准的2D平面培养的图像，随机异构表型分布。细胞染色为肌动蛋白（绿色），高尔基（橙色）和细胞核（蓝色）。比例尺= 100微米。对WT和II型肌球蛋白处理细胞平坦的表面上EC和（d）细胞（ 三 ）三维重建。比例尺= 20微米。880 / 51880fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51880/51880fig4.jpg" /> 图4:NIH3T3 研究高尔基体的表型上（ 一 ）平板和（b）EC细胞高尔基型分类的原理图和样品图像。细胞被归类为压实，这取决于α-值扩展或分散。 （ 三 ）高尔基表型的量化。比例尺= 10微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51880/51880fig5.jpg" /> 图5: <s仲&gt;研究NIH3T3核型的。（ 一 ）（左）的EC内部的真核细胞的共聚焦显微镜图像和染色肌动蛋白（绿色），高尔基（橙色）和细胞核（蓝色）。（右）的计划里面EC核方向。 （ 二 ）内乳油核的WT和（c）II型肌球蛋白处理过的细胞的角分布。 （ 四 ）对WT和II型肌球蛋白处理的细胞都为EC和核球形值的平面（P [WT EC -Blebb EC] &lt;0.001，P [Blebb 平 -Blebb EC] &lt;000.1; N WT 平 = 47，N WT EC = 94，N Blebb 平 = 59，N Blebb EC = 141细胞）。比例尺= 10微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图6: 现场固定样品和使用"eggcups"两个系统cytokinetic环的详细研究 （ 一 ）采用标准的2D 体外培养的cytokinetic环的时序。只有两个动蛋白（Lifeact-mcherry，红色）和肌球蛋白亮点（GFP标记，绿色）都在HeLa细胞卵裂沟（比例尺= 10微米）可见。 （ 二 ）时间关闭在HeLa细胞中的cytokinetic环的有丝分裂过程中使用"eggcups&#39;序列。的图像显示的肌动蛋白（红色）和肌球蛋白（绿色）。 "Eggcups"允许还是肌球蛋白积累的鉴定。一个例子是突出了一个箭头。 （比例尺= 5微米）。 （ 三 ）cytokinetic环也可以在裂殖酵母可视化。（左 ）细胞位于在一个平面上，所述CYTO<html>动力学环是仅作为两个点可见于"eggcups&#39;（右）的细胞:整个封闭件可以被捕获。肌动蛋白标记CHD-GFP（比例尺= 2微米）。时间分:秒。 （D - E）染色cytokinetic环的例子。表达HeLa细胞（ 四 ）肌动蛋白的GFP染色用于磷酸酪氨酸（PY），其也示出了在环（比例尺= 5微米）信号。表达GFP（ 五 ）HeLa细胞标记肌球蛋白和Lifeact-mcherry（肌动蛋白）的染色anillin。 Anillin显露在cytokinetic环和较少集中在皮质本地化。它显示共定位与肌动蛋白和肌球蛋白（比例尺= 5微米）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 80 / 51880fig7.jpg"/&gt; 图7:在&#39;eggcups&#39;到其它细胞类型和模型系统中的应用 （ 一个 ）U2OS（人骨肉瘤）。插图示出了分裂细胞。 （比例尺= 20微米）。 （ 二 ）的NIH3T3细胞表达GFP。差异表达水平可以容易地读出（比例尺= 20微米）。 （ 三 ）SW480细胞（比例尺= 20微米）。 （ 四 ）芽殖酵母;他们的周期时间是不变的。 （比例尺= 10微米）。 （ 五 ）C.线虫蠕虫;（左）在一个平面上（右）在&#39;eggcups&#39;，胚胎是从否则隐藏透视看到。 （比例尺= 10微米）。时间分:秒<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" fo:content-WIDTH ="5英寸"SRC ="/文件/ ftp_upload / 51880 / 51880fig8.jpg"/&gt; 图8: 在&#39;eggcups&#39;的阵列结构允许细胞群的自动分析 （ 一 ）的NIH3T3细胞在EC（比例尺= 20微米）。他们有GFP不同的表达水平。 （ 二 ）的单元位置的自动识别允许表达水平的个体分析。它总结了细胞群的GFP表达的柱状图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51880/51880fig8large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always"><tbody><tr><td> 模型系统 </td><td> 类型 </td><td> 培养基 </td><td> 观察中 </td><td> "eggcups"直径（μ;米） </td><td> 评论/说明 </td></tr><tr><td>哺乳动物细胞</td><td> NIH3T3 </td><td> 10％BCS高糖DMEM </td><td> 10％BCS的L-15 </td><td> 20 </td><td>其他的稳定细胞系，例如REF52或MDCK，以及原代细胞系，癌性细胞和/或干细胞还可以在"eggcups&#39;插入。 </td></tr><tr><td></td><td>海拉</td><td> 10％FCS的高糖DMEM </td><td> 10％FCS的L-15的</td><td> 25 </td><td>可以从很多不同的来源。 </td></tr><tr><td></td><td> U2OS </td><td> 10％FCS的高糖DMEM </td><td> 10％FCS的L-15的</td><td> 20-25 </td><td>可以从很多不同的来源。 </td></tr><tr><td></td><td> SW480 </td><td> 10％FCS的高糖DMEM </td><td> 10％FCS的L-15的</td><td> 17-20 </td><td>可以从很多不同的来源。 </td></tr><tr><td>酵母</td><td>裂殖酵母</td><td>洋菜板（YE5S）和液体介质（YE5S和EMM5S） </td><td>过滤消毒EMM介质（参见物料清单） </td><td>五</td><td>表面不需要与粘合剂蛋白官能化。 </td></tr><tr><td></td><td>芽殖酵母</td><td>琼脂平板（YPD）和液体介质（YEPD和SD） </td><td> SD存储介质</td><td>五</td><td>表面不需要与粘合剂蛋白官能化。 </td></tr><tr><td>胎</td><td> 线虫 </td><td> NGM板</td><td>超纯水</td><td> 25 </td><td>备选地M9培养基可用于长期实验。这种腌液的配方可以在这​​里找到:http://cshprotocols.cshlp.org/content/2009/5/pdb.rec11798.full?text_only=true </td></tr></tbody></table> 表 1: 针对不同的模型系统"eggcups&#39;文化条件。上述相关的协议可以很容易地适应通过只更换描述培养条件和&#39;eggcups&#39;的大小。

Watch this Scientific Journal Video about Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening at JoVE.com

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

我们报告的设备和新的方法来研究细胞和胚胎。单细胞精确下令微阵列。他们的3D坐月子是朝着在生理条件下遇到的3D环境的一个步骤，并允许细胞器的方向。通过控制细胞的形状，此设置最大限度地减少可变性中的标准试验的报道。

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

ordering-single-cells-single-embryos-3d-confinement-new-device-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.5K 次观看.  Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg. 我们报告的设备和新的方法来研究细胞和胚胎。单细胞精确下令微阵列。他们的3D坐月子是朝着在生理条件下遇到的3D环境的一个步骤，并允许细胞器的方向。通过控制细胞的形状，此设置最大限度地减少可变性中的标准试验的报道。 

视频: Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

科研

发育生物学

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

生物工程

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

使用自动高含量分析仪进行活细胞显微镜的果蝇胚胎制备和微注射

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

用于可重复模式形成的混合凝胶立方体器件中的初始三维单元群集控制

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

细菌细胞的三维成像，实现精确的细胞表现和精确的蛋白质定位

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...

免疫与感染

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

使用定量成像技术鉴定和表征异源细胞群体

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

生物学

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

单个细胞分辨率三维成像的有机体

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...