这项工作是支持的玛格丽特M. Alkek基金会RHF。

第1部分：台成立<ol><li>一股潮湿室是放置在塑料商会Kimwipe / DDH 2 O的准备。 </li><li>一个猎鹰管收集白蛋白，文化，戒指，表面玻璃和废物处置的菜都放在替补席上。 </li><li>耐热盘充满生理盐水1.4升（见注释[A]）。 </li></ol>第2部分：胚胎在盐水explanted <ol><li>鸡蛋是从孵化器中删除后16小时（第4阶段）。鸡蛋是由攻壳钳opene。壳牌件被删除。 </li><li>薄白蛋白收集猎鹰管。厚厚的白蛋白是用镊子除去。 </li><li>胚胎被放置在一个塑料盘内的盐水菜。剩下的白蛋白是用镊子除去。 </li></ol>第3部分：环中心的胚胎<ol><li>卵黄囊是用镊子倾斜，使胚胎朝上。 </li><li>卵黄囊是在赤道或以下程度的削减。 </li><li>卵黄膜是用细镊子，迅速去皮。卵黄膜为导向，因此，其颗粒的一面（非光亮）朝上。 </li><li>用细镊子，卵黄膜置于手表玻璃上。 </li><li>用细镊子，玻璃环适用于卵黄膜的顶部，并为中心的胚胎。 </li><li>卵黄膜是解除了周围的玻璃环的边缘。大会是从盐水菜。 </li></ol>第4部分：文化是设置在显微镜下<ol><li>卵黄膜是使用显微镜下细的镊子在玻璃环解除。 </li><li>使用巴斯德吸管，生理盐水从环的外缘。 </li><li>用细剪刀，多余的卵黄膜是从玻璃环的内侧边缘。护理是采取不刺破膜用吸管或镊子。 </li><li>胚胎是轻轻地用生理盐水冲洗，去除松散的膜和卵黄细胞。 </li><li> 200μL生理盐水添加到外缘环（这将有利于以后转让）。 </li><li>大会是覆盖着一个倒置的塑料盘。 </li></ol>第5部分：文化是转移到孵化器<ol><li>一个标记为“猎鹰培养皿。 </li><li>薄血白蛋白2.5毫升添加到猎鹰培养皿的底部。 </li><li> 200μL薄血白蛋白被添加到猎鹰培养皿的盖子内缘。 </li><li>倒菜是从大会。 </li><li>用细镊子，玻璃环下滑沿表玻璃的边缘，并转移到猎鹰菜。 </li><li>所有剩余的生理盐水是从环的内表面。 </li><li>猎鹰盘盖与盖和密封。 </li><li>大会是放置在潮湿的腔，然后在孵化器。 </li><li>胚胎培养24小时，在38 ° C </li></ol>第6部分：文化之后，胚胎是固定的，文化是转移到孵化器<ol><li>如果胚胎是要处理的原位杂交），一个固定的菜充满了冰冷的PBS（或DEPC - PBS。 </li><li>文化是从孵化器中删除，并立即置于冰上。立即充满了冰冷的PBS / DEPC - PBS的培养皿。 </li><li>胚胎从卵黄膜分离。胚胎转移到固定的菜，用巴斯德吸管的钝端。 </li><li>胚胎是使用罚款钳钝端牵制。 PBS覆盖固定盘被删除。固色剂添加（见注[B]）。 </li><li>根据应用，胚胎是固定的6-8小时，在4 °℃（恒温器），O / N在4 ° C（所在地），或在室温下1小时整装免疫组织化学</li><li>固定液用冰冷的PBS / DEPC PBS拆除和更换。 </li><li>对于下游应用原位杂交或immantibody染色，如：神经系统和心脏穿孔使用钝年底microcapillary针或显微切割刀，这将防止探头/抗体被困在后面的步骤。 注释 ：[A]盐水解决方案包括：（1升）：121.0 g氯化钠，氯化钾15.5克，10.4克氯化钙2 .2 H 2 O，12.7克氯化镁2 .6 H 2 O; B液（1升） ：2.4克钠2 HPO 4 .2 H 2 O，0.2克的NaH 2 PO 4 .2 H 2 O;高压灭菌器，在使用之前，混合120毫升与2700毫升H 2 O，加入180毫升乙混合[1]; [B]定影液是在使用前准备（4％PFA在所在地的PBS或DEPC - PBS）。 </li></ol>第7部分：代表结果文化之前，胚胎是在原始连胜阶段（HH4）。在文化时期的结束，胚胎发展到HH10（长2-3毫米），在培养皿的中心可见。标签carbocyanine荧光DII一组单元格，它是可能的前文化（0H），并按照他们的运动，在整个文化时期。在这种情况下，恒胜的节点（HN）以下细胞标记与DII。这些细胞被证明有助于逐步发展的体节（SOM）和脊索（N）。 <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" />第8部分：故障排除<table style="width: 698px; height: 241px;" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th>问题</th><th>原因</th><th>补救</th></tr><tr><td>胚胎仍然与蛋黄，而不是膜（步骤2） </td><td>卵子质量不佳</td><td>要求新鲜鸡蛋;在收到商店鸡蛋在13 ° C。孵育鸡蛋为入住日期的当天。 </td></tr><tr><td>卵黄膜滑离钟表匠的玻璃环以下的位置（步骤3） </td><td>白蛋白残余</td><td>在第2步，确保所有白蛋白是从膜拉离倾斜钳移除</td></tr><tr><td>胚胎是人迹罕至：位于下方的膜（步骤4） </td><td>膜错误的一边向上</td><td>在第3步，确保含有蛋黄颗粒膜的一侧朝上。的光泽，抛光的一面应朝下。 </td></tr><tr><td>淹没在盐水/白蛋白以下文化的胚胎（步骤6） </td><td>盐水/白蛋白文化前内环左;在膜孔</td><td>在第5步，确保所有白蛋白/生理盐水是从戒指内删除;在第4步，确保你不膜（使用钝结束钳钳皮尔斯） </td></tr><tr><td>胚胎解体以下文化</td><td>细菌感染</td><td>消毒所有的工具和玻璃器皿;使用抗生素/ antimycotic </td></tr></tbody></table>

<ol>
	<li>Pannett, P. A., Compton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+tissues+in+saline.">The cultivation of tissues in saline.</a> Lancet. 206, 381-384 (1924).</li><li>New, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+cultivation+of+the+chick+embryo+in+vitro.">A new technique for the cultivation of the chick embryo in vitro.</a> J. Embryol. Exp. Morph. 3, 326-331 (1955).</li><li>Alvarez, I. S., Araujo, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+in+whole+chick+embryo+cultures+by+FGF.">Neural induction in whole chick embryo cultures by FGF.</a> Dev. Biol. 199, 42-54 (1998).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+migratory+routes+of+primitive+streak+cells+in+the+chick+embryo.">Fates and migratory routes of primitive streak cells in the chick embryo.</a> Development. 122, 1523-1534 (1996).</li><li>Voiculescu, O., Papanayotou, C., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos.">Spatially and temporally controlled electroporation of early chick embryos.</a> Nature Protoc. 3, 419-426 (2008).</li></ol>

一个广泛的应用，从增长因素含珠 3，整个装载在原位杂交和整装免疫组织化学 4移植，可用于新的培养方法2。超过24小时期间的文化，使胚胎发育的连续监测中的应用，如时间的推移细胞运动分析5或绿色荧光蛋白含有电穿孔结构6监测。

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		<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>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish (2)</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker&#x2019;s glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Sharpening Stone Dan&#x2019;s Black Arkansas</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>62082-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microcapillary tube</td>
<td>Surgery</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>		</tbody>
		</table>
		</div>

method for culture of early chick embryos ex vivo (new culture)

鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，它为研究大脑，神经管，体节和心脏原基的形成和图案的理想工具。鸡胚培养的应用包括电的DNA或RNA的结构，以原位杂交和免疫组织化学分析基因的功能，生长因子，如涂珠FGFs和BMPs的移植，以及整装。本视频演示了在鸡胚培养的不同步骤，首先，胚胎是在生理盐水explanted。然后，胚胎中心的玻璃环上。周围的胚胎膜沿环的墙壁被取消。环，然后放置在文化菜含有白蛋白池。是密封的培养皿中，并放置在潮湿的腔，胚胎培养24小时。最后，胚胎是从环中删除，固定，并为进一步的应用处理。还介绍了故障排除指南。

Watch this Scientific Journal Video about Method for Culture of Early Chick Embryos ex vivo New Culture at JoVE.com

Method for Culture of Early Chick Embryos ex vivo New Culture

本视频演示了新的文化，鸡胚培养24小时以外的鸡蛋的方法。这种方法使研究的早期发展（原始连胜，以14索姆），鼠标E7 - 9对应一个时期。这项技术的应用，包括电，原位杂交和免疫组化。

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 ...

method-for-culture-of-early-chick-embryos-ex-vivo-new-culture

Research

JoVE Journal

Biology

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

15.5K 次观看.  Institute of Biosciences and Technology - Texas A&M Health Science Center. 本视频演示了新的文化，鸡胚培养24小时以外的鸡蛋的方法。这种方法使研究的早期发展（原始连胜，以14索姆），鼠标E7 - 9对应一个时期。这项技术的应用，包括电，原位杂交和免疫组化。

视频: Method for Culture of Early Chick Embryos ex vivo New Culture

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

科研

生物学

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

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 angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

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

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

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 ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

鼠类全胚胎培养方法，使用的肩型瓶培养系统

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

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 ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

在早期鸡胚跟踪形态组织变形

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

哺乳动物细胞培养，使用一种新的多层瓶中的比例增加

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

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 ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

切片培养法继牙胚在植文化的发展

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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 ...