이 작품은 RHF에 마가렛 M. 알켁 재단에 의해 지원되었다.

1 부 : 벤치 설정 <ol><li> 습기 챔버는 플라스틱 챔버에 Kimwipe / ddH 2 O을 삽입하여 준비가되어 있습니다. </li><li> 문화, 반지, watchglass 및 폐기물 처리에 대한 알부민, 요리를 수집하는 팰컨 튜브는 벤치에 게재됩니다. </li><li> Pyrex 요리는 1.4 리터의 호수 가득 (노트를 참조하십시오 [A]).입니다 </li></ol> 2 부 : 배아 호수에 explanted입니다 <ol><li> 계란 16 시간 (4 단계) 후 인큐베이터에서 제거됩니다. 계란은 집게로 껍질을 감청하여 opene 있습니다. 셸 조각이 제거됩니다. </li><li> 얇은 알부민은 팔콘 튜브에 수집됩니다. 굵은 알부민은 집게로 제거됩니다. </li><li> 배아는 생리 접시 안에 플라스틱 접시에 배치됩니다. 나머지 알부민은 집게로 제거됩니다. </li></ol> 파트 3 : 배아 반지에 중심 <ol><li> 그 배가 위쪽으로 얼굴 때문에 난황은 집게로 기울어져있다. </li><li> 난황은 적도 수준 또는 아래의 절단이다. </li><li> 좋은 집게를 사용하여 vitelline 막이 신속하게 벗겨 수 있습니다. vitelline 막은 그 세분화된 측면 (비 시니)가 이상 얼굴 너무 지향합니다. </li><li> 고급 집게를 사용하여 vitelline 막이 시계 유리에 배치됩니다. </li><li> 고급 집게를 사용하여 유리 반지가 vitelline 막 위에 적용되고 배아가 중심이다. </li><li> vitelline 막는 유리 반지의 가장자리 주위에 해제합니다. 조립은 살린 요리에서 제거됩니다. </li></ol> 4 부 : 문화 현미경으로 설정되어 <ol><li> vitelline 막는 현미경 미세 집게를 사용하여 유리 반지를 통해 해제됩니다. </li><li> 파스퇴르 피펫을 사용하여 호수는 반지의 바깥쪽 가장자리에서 제거됩니다. </li><li> 좋은 가위를 사용하여 초과 vitelline 막는 유리 반지의 안쪽 가장자리에서 제거됩니다. 관리는하지 피펫이나 집게로 막을 피어스로 이동합니다. </li><li> 배아는 부드럽게 풀어 세포막과 노른자의 세포를 제거하는 식염수에 씻어서 있습니다. </li><li> 200 μl 호수는 반지 (이것은 나중에 전송을 용이하게합니다)의 바깥쪽 가장자리에 추가됩니다. </li><li> 어셈블리가 거꾸로 플라스틱 접시로 덮여 있습니다. </li></ol> 제 5 부 : 문화 인큐베이터로 전송됩니다 <ol><li> 팰컨 문화 요리가 표시됩니다. </li><li> 얇은 알부민의 2.5ml은 팰컨 접시의 바닥에 추가됩니다. </li><li> 얇은 알부민의 200μl는 팰컨 접시의 뚜껑의 안쪽 가장자리에 추가됩니다. </li><li> 거꾸로 요리는 어셈블리에서 제거됩니다. </li><li> 좋은 집게를 사용하여 유리 반지 시계 유리의 가장자리를 따라 slided 및 팰컨 요리로 전송됩니다. </li><li> 모든 남아있는 호수는 반지의 안쪽 표면에서 제거됩니다. </li><li> 팰컨 요리는 덮개로 덮여과 봉인이다. </li><li> 조립은 습기 챔버에서 다음 인큐베이터에 배치됩니다. </li><li> 배아는 38 ° C.에 24 시간을 위해 양식 아르 </li></ol> 파트 6 : 다음 문화, 배 (胚)는 고정, 문화 인큐베이터로 전송됩니다 <ol><li> (배아는 현장 하이브리드화에 대한 처리 수있다면 또는 depc - PBS) 고정 요리는 얼음처럼 차가운 PBS로 가득합니다. </li><li> 문화 인큐베이터에서 제거하고 즉시 얼음에 위치합니다. 문화 요리는 즉시 얼음처럼 차가운 PBS / depc - PBS로 가득합니다. </li><li> 배아는 vitelline 막에서 분리됩니다. 배아는 파스퇴르 피펫의 뭉툭한 끝을 사용하여 고정 접시로 전송됩니다. </li><li> 배아는 무딘 종단 미세 집게를 사용하여 넘어뜨린 것입니다. 고정 접시를 덮고 PBS가 제거됩니다. 정착액가 추가됩니다 (노트를 참조하십시오 [B]). </li><li> 응용 프로그램에 따라, 배아는 4에서 6-8 시간에 대한 고정 ° C (그라 이오 스탯), 4 O / N ° C (시터스)를, 또는 전체 탑재 immunohistochemistry에 대한 RT에서 1 시간. </li><li> 정착액은 얼음처럼 차가운 PBS / depc PBS로 제거하고 교체합니다. </li><li> 원위치 하이브리드화 또는 immantibody의 얼룩에 같은 다운 스트림 응용 프로그램의 경우 : 신경계와 심장이 뭉툭한 끝 microcapillary 바늘이나 microdissection 나이프를 사용하여 천공되며 이것은 나중 단계에서 프로브 / 항체의 트래핑 방지할 수 있습니다. 참고 : [A] 호수가 구성되어 솔루션 (1 게 ...) : 121.0 g NaCl, 15.5 g KCl, 10.4 g CaCl 2 0.2 H 2 O, 12.7 g MgCl 2 0.6 H 2 O, 용액 B (일리터에 대한) : 2.4 g 나 2 HPO 4 0.2 H 2 O, 0.2 g 아니 2 PO 4 0.2 H 2 O, 압력솥, 사용하기 전에는, 120 ML를 섞어 2700 ML H 2 O로, 180 ML B. 혼합 추가 [1]; [B] 정착액이 (4 % 시터스에 대한 PBS 또는 depc - PBS에 PFA)를 사용 직전 준비가되어 있습니다. </li></ol> 7 부 : 대표 결과 문화 이전의 배아는 원시 리크 단계 (HH4)에 있습니다. 문화 기간의 끝에, 배아는 HH10 (길이 2-3밀리미터)을 개발하고 문화 요리의 중앙에 표시됩니다있다. 형광 DiI를 carbocyanine과 세포의 그룹을 분류하는 것이 가능단지 문화 전 (오)와 문화 시대를 통해 자신의 움직임을 따르십시오. 이 경우, Hensen의 노드 (HN) 아래의 세포는 DiI으로 표시되었습니다. 이러한 세포는 점차적으로 개발 somites (솜)와 notochord (N)에 기여 표시됩니다. <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" /> 8 부 : 문제 해결 <table style="width: 698px; height: 241px;" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th> 문제 </th><th> 원인 </th><th> 치료제 </th></tr><tr><td> 난자는 멤브레인 (2 단계)로 내려 와요 대신 노른자로 남아 </td><td> 불쌍한 계란 품질 </td><td> 접수시 13 스토어 계란 ° C; 요청 신선한 계란을 생산. 계란에게 도착 날짜와 같은 하루를 품어. </td></tr><tr><td> 멀리 반지 시계 제조인의 유리 아래 배치 (3 단계)에서 Vitelline 막 슬라이드 </td><td> 알부민 잔존물 </td><td> 2 단계에서는 모든 알부민이 기울어져 집게로 막은에서 돌아서에 의해 제거되어 있는지 확인합니다 </td></tr><tr><td> 난자는 액세스할 수 있습니다 멤브레인 (4 단계) 아래에 자리잡고 </td><td> 막의 잘못된 측면이 이상합니다 </td><td> 3 단계에서 노른자의 과립이 들어있는 막의 측면이 위쪽을 향하고 있는지 확인하십시오. 반짝, 광택 쪽을 아래쪽으로 직면한다. </td></tr><tr><td> 난자는 (단계 6) 호수 / 알부민 다음과 문화에 빠져들 </td><td> 이전 문화에 반지 안쪽 왼쪽 알부민 / 살린, 막에 구멍 </td><td> 5 단계에서 호수 / 모든 알부민은 반지 안쪽에서 제거되었는지 확인, 4 단계에서 (퉁명스러운 종료 포셉 사용) 포셉와 피어스 막하지 않도록 </td></tr><tr><td> 배아는 다음과 문화를 분해 </td><td> 세균 감염 </td><td> 모든 도구 및 유리 제품을 소독하는 것은, 사용 antimycotic / 항생제 </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>

새로운 문화 방법 2 원위치 하이브리드화 및 전체 마운트 immunohistochemistry 4 전체 마운트 구슬 3, 포함하는 성장 인자의 이식에 이르기까지 애플 리케이션의 광범위한 사용하실 수 있습니다. 24 시간 기간 동안 문화는 시간 저속 세포의 움직임 분석 5 GFP 6 electroporated 구조를 포함하는 모니터링과 같은 어플 리케이션에서 배아 개발의 지속적인 모니터링이 가능?...

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

여자의 난자는 초기 배아 발달의 연구에 귀중한 도구입니다. 투명성, 접근성 및 조작 용이성, 그건 뇌, 신경 튜브, 체절과 심장 primordia의 형성과 patterning을 공부를위한 이상적인 도구가합니다. 여자는 배아 문화의 응용 프로그램은 현장 하이브리드화과 immunohistochemistry의 유전자 기능 등 FGFs 및 BMPs와 같은 성장 인자 코팅 구슬 이식뿐만 아니라 전체 마운트를 분석하기 위해 DNA 또는 RNA 구조의 electroporation을 포함합니다. 이 비디오는 여자의 배아 문화의 여러 단계를 보여주는, 첫째, 배아는 호수에 explanted 있습니다. 그런 다음, 배 (胚)는 유리 반지를 중심으로합니다. 배아를 둘러싼 세포막은 반지의 벽면을 따라 해제됩니다. 반지는 다음 albumine의 수영장이 포함된 문화 접시에 배치됩니다. 배아는 최대 24 시간까지 교양있는 곳을 문화 요리는 밀봉하여 습기가 실내에 배치됩니다. 마지막으로, 배아는 반지에서 제거 고정 더욱 응용 프로그램에 처리됩니다. 문제 해결 가이드도 제공됩니다.

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

이 비디오는 새로운 문화, 병아리의 배아는 최대 24 시간까지 계란 이외의 양식하는가하는 방법을 보여줍니다. 이 방법은 초기 개발 (14 솜으로 원시적인 승리.) 마우스 E7 - 9에 해당하는 기간 동안 공부를 하나 있습니다. 이 기술의 응용은 현장 하이브리드화과 immunohistochemistry에 electroporation을 포함합니다.

조기 칙 태아 예 생체내 (새로운 문화)의 문화에 대한 방법

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

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Biology

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

15.5K 조회수.  Institute of Biosciences and Technology - Texas A&M Health Science Center. 이 비디오는 새로운 문화, 병아리의 배아는 최대 24 시간까지 계란 이외의 양식하는가하는 방법을 보여줍니다. 이 방법은 초기 개발 (14 솜으로 원시적인 승리.) 마우스 E7 - 9에 해당하는 기간 동안 공부를 하나 있습니다. 이 기술의 응용은 현장 하이브리드화과 immunohistochemistry에 electroporation을 포함합니다.

비디오: 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.

조기 칙 태아의 현장 하이브리드화에서 더블 전체 산

연구

생물학

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.

병아리 예 비켜 문화와 예 비켜 CAM 어세이 : 그게 정말로 작동하는 방법

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.

에서 Organotypic Murine 망막 Wholemount 문화 생체내 같은

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.

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

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.

기본 인간 난관 상피 세포의 전의 VIVO 문화

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.

초기 여자는 배아에서 Morphogenetic 조직 Deformations 추적

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.

새로운 Multilayered 플라스크를 사용하여 포유류 세포 배양의 스케일 업

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.

소설<em&gt; 생체</em배아 마우스 사랑을&gt; 문화 방법

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.

이식편 문화 치아 세균의 개발을 다음과 같은 사항에 대해 슬라이스 문화 방법

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.

마우스 배아의 피부와 Melanoblast 이주의 라이브 영상의 생체 문화

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