作者想感谢优秀的技术援助和教授Ruebben J. Kueper J. Wittschier慷慨地提供肿瘤物质。

所有的设备和试剂必须购买无菌或需要加热或蒸汽消毒或用70％乙醇消毒。 作者状态，对动物的实验是根据欧洲共同体理事会指令（86/609/EEC），美国国立卫生研究院的机构动物所规定的实验程序和法规的动物护理和使用准则保养和使用委员会（IACUC）在杜伊斯堡 - 埃森大学（德国）。 第1部分：蛋孵化<ol><li>受精卵可存储长达一个星期在13 ° C。 </li><li>鸡蛋孵育72小时，躺在水平的孵化器与移动的托盘，旋转的鸡蛋12次，每天不断。湿度保持在60 - 62％，孵化温度为37.5 ° C。 注：在开始孵化，灰尘，羽毛和粪便，小心取出从鸡蛋壳机械常见，灰色锯齿手纸巾，其中有一个柔软的表面结构，而不是粗糙的干擦。用乙醇或消毒擦拭的鸡蛋，但是，显着降低胚胎成活率不论所使用的液体。 </li></ol>第2部分： 防爆OVO文化<ol><li>经过72小时的孵化，蛋是从孵化器中删除。 注 ：卵孵育72小时，因为-根据我们的实验-原因不明，但在与奥尔巴赫等al.1早期发展阶段的协议生存率有显着降低。黄麻袋，这是由凸轮尚未覆盖的阶段最迟72小时变得更薄，往往要坚持领导（一）少量出血，在坚持和（ii）破裂的蛋黄，一袋鸡蛋壳膜。因此提出的方法并不适用于年龄超过72小时的胚胎。 </li><li>因为胚胎在一定程度上抵抗旋转的鸡蛋的顶部，胚胎的所在，用铅笔标记。 </li><li>鸡蛋是水平之上的铅笔标记，并破获一个80毫米的三角磁力搅拌铺设面向鸡蛋的长轴垂直栏的边缘。 注 ：更好的感觉，没有应使用手套，但双手应消毒，用70％乙醇。重要的是，开辟了鸡蛋壳，以及卵膜。蛋清泄漏是一个安全标志，卵膜已穿孔。 </li><li>鸡蛋是保持着密切的培养皿底部的蛋清，以避免进一步的泄漏。 注：在最初开放的过程中，重要的是在培养皿的底部，鸡蛋白是这个结果仍然连接到蛋内的其余部分，在一个拥有顶级内的胚胎蛋黄蛋内的真空鸡蛋。通过手指的压力，控制进气口进入卵子或解除卵子，从而增加了连接蛋清即真空列，可调节的真空。 </li><li>轻轻施加压力，通过裂缝和鸡蛋拇指和中指的赤道运行经络是可以调节的真空。蛋的内容可以被转移到培养皿中完好无损，如果与蛋黄的鸡蛋轻轻地抬起，仔细分离两半壳。胚胎和卵黄船只应趴在一个完好的蛋黄顶部。 注意 ：如果不保持顶部铅笔标记，开裂的外壳时，可能会伤害胚胎。如果鸡蛋没有破解足够仔细或胚胎72小时以上（见上文），黄麻袋枝壳和经常破裂。同样的情况时，孵化器的湿度不低于60％或鸡蛋在孵化过程中旋转。 </li><li>返回前OVO文化的孵化器，并保持在37.5 ° 38.2 ° C和60％的湿度。 CO 2或O 2的供应是没有必要的，如果孵化器是不是封闭的密封（通风电网局部开放！），并使用特殊的Petri与盖和盘之间的间隔菜。 注 ：移动的胚胎，应保持在孵化的第一天到最低限度。在高死亡率的太多躁动的结果。从孵化第9天起，胚胎不敏感鼓动。 死胚应立即删除，以避免感染。 如果使用细胞培养孵化器，密封封闭，O 2的供应是不会死的胚胎强制性。湿度是必不可少的CAM不是干出来的，如果孵化温度超过37 ° C，胚胎会发展得太慢。 </li></ol>第3部分：物质的血管生成实验中的应用<ol><li>蒸压折叠滤纸（5由打孔器打孔，直径0.5毫米），用于支持应用到CAM的液体物质的材料（载体）。 注意 ：几乎每个载体材料（见下表）引起的CAM刺激，尤其是在孵化第10天前申请，因为它改变了发展中国家的CAM血管结构。这也包括潜在的惰性物质，如羟乙基纤维素箔，聚四氟乙烯（PTFE - pledgets）的海绵，胶原蛋白海绵，胶原蛋白的金属箔，用无菌纱布（拭子），圆形盖玻片，和硅环，而这一切导致假阳性结果。滤纸似乎导致至少刺激性，因此，在我们的实验中使用。 </li><li>当凸轮的全面发展，多达6种不同的样本可能会被应用，其效果可以在一个单一的凸轮相比。 </li><li>为了避免干燥过滤器额外的剂量（3微克本土angiotropin）或缓冲液（5μLPBS）应重新申请每天。 </li><li>从申请后第3天，血管定位对血管生成物质的过滤器，而周边控制的血管格局不变。 注：CAM的不应该误认为蛋黄，麻袋。 CAM是一个动态发展的结构，而蛋黄麻袋在开发过程中有一点差异的静态。发展日间三个发展中国家的CAM可以确认胚胎的尾部结束第一次作为一个小囊泡。初步囊泡之间的第三天，每天十个扩展，创建一个折叠的两层膜overgrowing黄麻袋的速度越来越快。申请地点应半之间的胚胎和CAM（境）倍的方式，否则胚胎阻碍在透射光显微镜下观察效果。另外，与血管生成物质的过滤器是内在的，当凸轮不断增加。 </li></ol>第4部分：细胞接种到CAM 细胞4.1.Preparation的<ol><li>聚合细胞从培养瓶转移到鹰管，使用25毫升无菌塑料吸管使用吸管援助。 </li><li>估计细胞数，15μL的细胞悬液转移到一个纽鲍尔室和细胞在显微镜下手工计算。 </li><li>猎鹰管离心3分钟，在室温下在1000 RPM和丢弃上清液。 </li><li>对于活细胞的跟踪，细胞用PBS稀释至所需浓度（此处为：2 × 10 7细胞/ ml），如PKH26（Sigma公司），一个红色荧光活细胞膜标记试剂盒染色，根据制造商的协议。 细胞重新悬浮在细胞培养液（优先贝科的修改鹰的介质; DMEM培养液）注射所需浓度（10 7细胞/毫升）或接种（3X10 6细胞/毫升）。根据文献15-17日 ，我们的经验已经出版的浓度，不同的细胞浓度用于注射与接种。 </li></ol> 4.2接种细胞到CAM <ol><li>使用锋利的手术刀，戒指（〜1mm厚）从一个1毫升枪头或周边静脉导管（PVL）管被切断，适用于E6 - E14鸡胚培养前 OVO CAM 。 注意 ：尽量不要“看到”了几次，但枪头或用导管穿过切割使用一个新的，非常尖锐的手术刀锋利的边缘，以避免上环。检查微裂缝产生的尖锐的边缘，在显微镜下环。 </li><li>根据环的大小20 3X10 6细胞/ ml的细胞液吸管在共成一个圈，或分割成多个环卷。 注 ：在某些情况下，轻轻地刮取CAM性伤痛的一个先决条件，例如，以方便接种细胞17 capillarisation移植（见下文）。但是，如果性伤痛的CAM是不打算或contraindicative，如肿瘤细胞的侵袭潜能的研究，戒指不应该被用于接种，但应直接应用于环或环细胞本身可能诱发微病变。如果使用环，这些应该削减尽可能薄，以尽量减少重量和避免的CAM缩进。 </li></ol>第5部分：玻璃体内注射细胞<ol><li>汉密尔顿微升注射器，70％的乙醇，最后用无菌PBS冲洗数次。 </li><li>充满了微量注射器的准备染色或不染的细胞悬液（见4.1部分）。 注：注射部位，要慎重选择，不要伤害比用针穿透血管的胚胎（brain!）的CAM或相邻结构。注射应根据解剖双眼。 </li><li>显微镜下控制小心，但很快用注射器针头穿透CAM和眼睛层，并不断注入10最大20μL细胞悬液（优先PBS稀释）进入玻璃体鸡胚培养前 OVO 。 注意：如果不能用针穿透，在CAM，它可能会用两个镊子轻轻撕裂它除了在网站很少或根本没有血管中删除。 </li><li>针应保持1-2秒，进入眼内注入细胞悬液，以避免因漏电损失。 </li></ol>第6部分：夹层鸡肢芽<ol><li>鸡蛋是从孵化器，孵化后3〜4天（E3/E4 =汉堡汉密尔顿（HH）stage18 - 23） </li><li>鸡蛋裂开了一个三角磁力搅拌棒的边缘上，转移到培养皿中（详情：请参阅第2部分），并从连接的蛋黄血管解剖，胚胎切割一个圆圈，用弹簧剪刀。 </li><li>胚胎转移到一个新的充满冷，无菌PBS的培养皿中，用一个小漏勺洗净，一种躁动。 </li><li>胚胎被转移到一个小培养皿中，用无菌PBS填充，从周围的膜中解脱出来，用细镊子小心撕裂他们分开。 </li><li>肢芽脱离显微镊子尽可能靠近身体，掌握和转移到一个主机8 - 10日龄的鸡养殖前 OVO（见嫁接过程）CAM 。 </li></ol>第7部分：制备小鼠肢芽<ol><li>一次怀孕交配设立和指定的女性交配检测阴道塞的一天上午妊娠0天。 </li><li>怀孕的女性是牺牲颈椎错位时，胚胎的发展达到了胚胎一天（五）10至13，并固定于蜡板上。 </li><li>腹壁蘸70％乙醇，沿中线切开皮瓣是固定的横向引脚。 </li><li>子宫的两个角是从腹部，用冷PBS分离并转移到烧杯中删除。 </li><li>胚胎分离，转移到培养皿和子宫壁，并使用镊子仔细胚胎膜被删除。 </li><li>肢芽切断使用弹簧剪刀或罚款钳分离尽可能靠近身体，掌握和转移的CAM 8 - 10日龄主机鸡养殖前OVO（见嫁接过程）。 </li></ol>第8部分：收集的肿瘤样本<ol><li>肿瘤活检收集在2毫升的Eppendorf管洋溢着肿瘤细胞的特定的培养基（在这里：Leibovitz的培养基），并在室温下保存。 </li><li>肿瘤活检无菌手术刀切成1-2万立方米件。 注意 ：如果肿瘤样本太小，他们可能不会附加到CAM，如果过大，可能会受伤，在CAM，胚胎可能死亡。 </li><li>一块从活检样品的核心是嫁接到CAM的8 - 10日龄主机鸡养殖前 OVO（见嫁接过程） 。 注 ：以活检表面的材料，增加肌肉或结缔组织污染的风险。 </li></ol>第9部分：嫁接过程<ol><li>肢芽或肿瘤标本，充分开发的8 - 10日龄鸡胚培养的前 OVO使用CAM嫁接。 </li><li> Ÿ附近的血管分叉CAM移植占尽地利。 </li><li>在所需的应用程序的网站，CAM是有选择性的创伤轻轻地刮取关闭（钳）双上皮细胞层上部peridermal部分，留下完整的基底层。 注 ：这是必不可少的，以避免出血或可见毛细血管破裂。 </li><li>移植物转移到CAM附加最小的PBS。根据嫁接，它可能是一次或两次放置在一个网站的CAM，在没有最终嫁接的目的是清除过多的PBS，然后终于在准备好的精神创伤的CAM网站嫁接。 </li><li>抓住罚款钳移植到CAM分层。根据移植物的敏感性，轻的压力可能被应用（不适合肢芽）机动嫁接成的CAM缩进（适合肿瘤样本） 。 注意 ：由于雏鸡免疫系统的发展，在胚胎一天（五）18 OVO异种移植前的文化不应该超越E17的延长主机鸡胚经常死。 有可用的主机凸轮，分别培养鸡胚前OVO，定时，以配合您所选择的捐助者组织的来源是很重要的。 </li></ol>第10部分：重新嫁接<ol><li>主机鸡胚被杀害斩首。 </li><li>与CAM删除附加的移植是一个圆形切割指出剪刀，并转移到培养皿中，用PBS充满。 </li><li>过度CAM是减少弹簧剪刀，从移植前附着部位除外。 </li><li>更大的肿瘤样本的情况下，削减一半或几个较小的部分移植和面临的第二个主机的新的CAM与切割边缘重新嫁接。 </li></ol>第11部分：代表结果<img src="/files/ftp_upload/1620/1620fig1.jpg" alt="图1" /> 图1。无壳的鸡文化 鸡胚不同发展阶段的培养在培养皿中的前 OVO 。 <img src="/files/ftp_upload/1620/1620fig2.jpg" alt="图2" /> 图2 前OVO CAM血管生成试验 受精鸡蛋中培养水平在37℃，湿度为60 - 62％，并在胚胎第4天（E4）培养皿破获。在同等条件下继续培养。在E10的无菌滤纸（直径5.5毫米）是分层的CAM上，浸泡5μLPBS或3微克本土angiotropin6。在E14，毛细血管呈明显的调整对angiotropin浸泡过的滤纸。 <img src="/files/ftp_upload/1620/1620fig3.jpg" alt="图3" /> 图3。EX OVO肢芽嫁接 鸡（E3/E4）和鼠标（E13）的肢芽嫁接到壳鸡文化CAM。从CAM的血管进入后两天的肢芽。肢芽的发展， 继续在这些前OVO文化在鸡达到两指骨阶段，并显示在小鼠肢芽趾间网减少。 <img src="/files/ftp_upload/1620/1620fig4.jpg" alt="图4" /> 图4 前OVO接种肿瘤样本。 一个膀胱癌的活检样本接种到10日龄的鸡胚培养前OVO CAM。经过两天在文化，连接到CAM血管肿瘤标本后7天，观察多个血管进入移植。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chorioallantoic+membrane+assay:+vascularized+3-dimensional+cell+culture+system+for+human+prostate+cancer+cells+as+an+animal+substitute+model.">Chorioallantoic membrane assay: vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model.</a> J. Urol. 166, 1502-1507 (2001).</li><li>Hall, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafting+of+organs+and+tissues+to+the+chorioallantoic+membrane+of+the+embryonic+chick.">Grafting of organs and tissues to the chorioallantoic membrane of the embryonic chick.</a> TCA Manual. 4 (3), 881-883 (1978).</li><li>Kunzi-Rapp, K., Kaskel, P., Steiner, R., Peter, R. U., Krahn, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+blood+levels+of+human+S100+in+melanoma+chick+embryo+xenografts'+circulation.">Increased blood levels of human S100 in melanoma chick embryo xenografts' circulation.</a> Pigment Cell Res. 14, 9-13 (2001).</li><li>McFall, R. C., Sery, T. W., Makadon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+new+continuous+cell+line+derived+from+a+human+retinoblastoma.">Characterization of a new continuous cell line derived from a human retinoblastoma.</a> Cancer Research. 37, 1003-1010 (1977).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+effect+of+matrix+metalloproteinase+down-regulation+on+vascular+intravasation+and+metastasis+of+human+fibrosarcoma+cells+selected+in+vivo+for+high+rates+of+dissemination.">Unexpected effect of matrix metalloproteinase down-regulation on vascular intravasation and metastasis of human fibrosarcoma cells selected in vivo for high rates of dissemination.</a> Cancer Research. 65, 10959-10969 (2005).</li><li>Kim, J., Kovalski, K., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=requirement+for+specific+proteases+in+cancer+cell+intravasation+as+revealed+by+a+novel+semiquantitative+PCR-based+assay.">requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay.</a> Cell. 94, 353-362 (1998).</li></ol>

创新或只是另一个小鸡文化协议？ 1-4与前壳的文化协议鸡胚的总生存率分别为低，例如CA。 30％1。此视频文章中所述的成品前OVO文化协议，相反，允许超过50％的生存率。 OVO文化经典相比前鸡胚的 OVO文化，大大方便了CAM和鸡胚的无障碍，使他们的实验操作和连续监测。这种文化的方法，可用于各种广泛的应用，增强血管生成</s...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fertilized Eggs</td>
<td>Animal</td>
<td>S&ouml;ries Trockels, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator Type 3000 with rotating egg tray</td>
<td>Tools</td>
<td>Siepmann GmbH Germany</td>
<td>9503</td>
<td>Maintain at 37&deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>Egg incubator BSS 160</td>
<td>Tools</td>
<td>Grumbach, Germany</td>
<td>8101</td>
<td>Maintain at 37-387deg;C with relative humidity set above 60%</td>
</tr>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile
</td>
</tr>
<tr>
<td>Dulbecco's modified eagle's medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 6429</td>
<td>DMEM culture medium</td>
</tr>
<tr>
<td>Leibovitz's L-15 Medium</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>31415-029</td>
<td>here: collection medium for tumor samples</td>
</tr>
<tr>
<td>Tumor cell -specific medium</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>each research group should use the medium they culture their tumors cells in or common culture media, e.g. Leibovitz's Medium or Dulbecco's modified eagle's medium supplemented with 15% fetal bovine serum (FBS), 4mM L-glutamine and 50 &mu;M insulin</td>
</tr>
<tr>
<td>70% EtOH</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic stir bar, triangled 80 mm</td>
<td>Tool</td>
<td>VWR</td>
<td>442-0391</td>
<td>A triangled magnetic stir bar serves well to crack the eggs shell.</td>
</tr>
<tr>
<td>Forceps DUMONT #5 </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors, straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Fine Iris scissors, straight</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14094-11</td>
<td>Use to cut our the CAM</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>microliter syringe 1702 TLLX, 25 &mu;l</td>
<td>Tool</td>
<td>Hamilton</td>
<td>80222</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Hamilton 33-G needle (15 mm)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>18073-15</td>
<td>Use for injection into the vitreous</td>
</tr>
<tr>
<td>Sterile scalpels</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use for dissecting tumor samples</td>
</tr>
<tr>
<td>Small drain spoon</td>
<td>Tool</td>
<td>Geuder, Germany</td>
<td>15758</td>
<td>Use to transfer of small chick embryos</td>
</tr>
<tr>
<td>Wax board with fixing pins</td>
<td>Tool</td>
<td>Self made</td>
<td>&nbsp;</td>
<td>Use to fix of animals for dissection</td>
</tr>
<tr>
<td>Petri dishes 60 x 15 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>628102</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 92 x 16 mm</td>
<td>Tool</td>
<td>Sarstedt</td>
<td>82.1472</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Petri dish 100 x 20 mm</td>
<td>Tool</td>
<td>Greiner</td>
<td>664160</td>
<td>extra deep, with spacers for ventilation</td>
</tr>
<tr>
<td>Beaker</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>300-600 ml</td>
</tr>
<tr>
<td>FALCON tubes</td>
<td>Tool</td>
<td>FALCON</td>
<td>&nbsp;</td>
<td>15 ml and 50 ml</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>1.5 ml and 2 ml</td>
</tr>
<tr>
<td>1ml pipette tips</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use to cut plastic rings for application of substances / cells</td>
</tr>
<tr>
<td>Peripheral venous catheter (PVL)</td>
<td>Tool</td>
<td>Viggo&reg;</td>
<td>&nbsp;</td>
<td>Use to cut silicon rings from the tube for application of substances / cells</td>
</tr>
<tr>
<td>Pipettes and tips</td>
<td>Tool</td>
<td>Eppendorf / Abimed</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Autoclaved folded Filters 595 1/2, 110 mm diameter</td>
<td>Tool</td>
<td>Schleicher &amp; Schuell</td>
<td>311643</td>
<td>Carrier, which caused least irritation of the CAM; used in the paper</td>
</tr>
<tr>
<td>PTFE-Pledget, non resorbing, 6,3 x 152.4 x 1.6 mm</td>
<td>Tool</td>
<td>santec-medical</td>
<td>REF 277, LOT 832/511-1</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Hxdroxyethylcellulose Tylose H 100000</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Carrier mixed with Kollidon 17 PF; caused false positive results</td>
</tr>
<tr>
<td>Kollidon 17 PF</td>
<td>Reagent</td>
<td>BASF</td>
<td>S30200</td>
<td>mixed with Hydroxyethylcellulose; caused false positive results</td>
</tr>
<tr>
<td>Collagen Biomatrix TissuDura</td>
<td>Tool</td>
<td>Baxter Healthcare S.A.</td>
<td>REF B2246000999999</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Round glass cover slips, 11 mm</td>
<td>Tool</td>
<td>Assistent Germany</td>
<td>1001/0011</td>
<td>Carrier; causes false positive results</td>
</tr>
<tr>
<td>Bovine Collagen Sponge</td>
<td>Tool</td>
<td>Wyeth</td>
<td>&nbsp;</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Resodont Absorbable Collagen Membrane</td>
<td>Tool</td>
<td>Resorba Woundcare Germany</td>
<td>LOT 280303</td>
<td>Carrier; caused false positive results</td>
</tr>
<tr>
<td>Non-Woven Swabs</td>
<td>Tool</td>
<td>Fink &amp; Walter GmbH</td>
<td>REF 328132</td>
<td>Carrier; caused false positive results</td>
</tr>		</tbody>
		</table>
		</div>

chick ex ovo culture and ex ovo cam assay: how it really works

在繁殖初期的鸡蛋之间在体外和体内系统，并提供了血管的测试环境，不仅要研究血管生成，但更要研究肿瘤的发生。后的鸡胚绒毛尿囊膜（CAM）的发展，其血管网络可以轻松地访问，操纵和观察，因此对血管生成的实验提供了一个最佳设置。由于淋巴系统没有完全开发的后期阶段，直到孵化，鸡胚作为一个能够维持无特定物种的限制嫁接的组织和细胞的自然免疫缺陷的主机。除了培育发展异体和异种移植，CAM血管网络提供了一个独特的支持性环境对肿瘤细胞的血管内，传播和血管逮捕和被逮捕细胞渗出，形成微转移灶的一个仓库里。 实验目的，在最近期的研究CAM暴露切割通过蛋壳的一个窗口，并进行了实验在 OVO无障碍CAM和效果的观察和照片文件的可能性，造成很大的局限性。当鸡胚无壳培养使用1-4，提供了无实验细节，如果在所有出版，这些文化的存活率低。我们精制的鸡胚前OVO文化的方法显着引进的鸡蛋含量的合理控制挤压。这些当然OVO文化，加强无障碍CAM和鸡胚，使容易在体内影响的文件，并促进胚胎的实验操作。这使得大量的科学问题进行了成功的应用：（1）作为改善血管生成的实验5,6，（2）实验设立便利注射在鸡胚眼7-9的玻璃体，（3）作为一个测试环境中传播和血管内的分散维持移植成功和肢芽鸡和小鼠13以及文化作为一种改进系统 ​​肿瘤细胞从细胞接种于CAM 10-12线，（4（5）嫁接，繁殖，重新嫁接坚实的原发肿瘤组织活检获得表面上的CAM 14）为。 在这个视频文章中，我们描述了建立一个精致的小鸡前OVO文化和CAM与存活率的检测，50％以上。除了 ​​我们提供了一个一步一步前OVO文化的科学应用的大量成功应用的示范。 丹尼尔Dohle，苏珊D.婆娑，和塞巴斯蒂安Gustmann同等贡献这项研究。

Watch this Scientific Journal Video about Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works at JoVE.com

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

鸡胚绒毛尿囊膜（CAM）是一个独特的，自然免疫缺陷的支持文化环境研究血管生成和肿瘤发生。此视频文章演示了在鸡的不同步骤<em&gt;前OVO</em&gt;企业文化，潜在的血管生成的物质和成功接种肿瘤细胞和组织上的CAM表面的应用程序。

小鸡前OVO文化前OVO CAM含量：它是如何真正起作用

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

chick-ex-ovo-culture-and-ex-ovo-cam-assay-how-it-really-works

Research

JoVE Journal

Biology

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

48.7K 次观看.  University of Duisburg-Essen. 鸡胚绒毛尿囊膜（CAM）是一个独特的，自然免疫缺陷的支持文化环境研究血管生成和肿瘤发生。此视频文章演示了在鸡的不同步骤前OVO企业文化，潜在的血管生成的物质和成功接种肿瘤细胞和组织上的CAM表面的应用程序。

视频: Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

利用鸡胚神经管破译基因定义的组的神经元轴突途径在OVO电穿孔

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

在OVO在鸡胚视网膜电穿孔

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

基因转移到老年人的鸡胚<em&gt;前OVO</em&gt;电穿孔

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

小鸡前OVO文化前OVO CAM含量：它是如何真正起作用

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