Bu çalışma, Margaret M. RHF Alkek Vakfı tarafından desteklenmiştir.

Bölüm 1: Tezgah kurmak <ol><li> Nemli odasına plastik odasında Kimwipe / GKD 2 O koyarak hazırlanır. </li><li> Falcon tüp albümin, kültür, yüzük, Dürbün ve atık bertarafı için yemekleri toplamak için bir tezgah üzerine yerleştirilir. </li><li> Kabına 1.4 lt tuzlu su ile dolu (notlara bakınız [a]). </li></ol> Bölüm 2: Embriyo tuzlu eksplante <ol><li> Yumurta, 16 saat sonra (evre 4) inkübatör kaldırılır. Yumurta kabuk forseps ile dokunarak opene. Shell adet kaldırılır. </li><li> Ince albümin Falcon tüp toplanır. Kalın albümin forseps ile kaldırılır. </li><li> Embriyo tuzlu çanak içinde plastik bir tabak içinde yer alır. Kalan albumin forseps ile temizlenebilir. </li></ol> Bölüm 3: Embriyo halka merkezli <ol><li> Yolk kesesi, embriyo yukarı bakacak şekilde forseps ile hareket ettirildiğinde. </li><li> Yolk kesesi ekvator düzeyinde veya altında kesilir. </li><li> Vitellin membran ince forseps kullanarak, hızlı bir şekilde soyulur. Vitellin membran granüler tarafında (parlak ama) yukarı bakacak şekilde aydınlatmaktadır. </li><li> Vitellin membran ince forseps kullanarak, saat camı üzerine yerleştirilir. </li><li> Ince forseps kullanarak, bir cam yüzük vitellin membran üstüne uygulanır ve embriyo ortalanır. </li><li> Vitellin membran cam halka kenarlarında kaldırdı. Montaj tuzlu çanak kaldırılır. </li></ol> Bölüm 4: kültür mikroskop altında kurulur <ol><li> Vitellin membran cam, mikroskop altında ince forseps kullanarak halka üzerinden kaldırdı. </li><li> Pasteur pipeti kullanarak, tuzlu su, dış ring kenarından kaldırıldı. </li><li> Ince makas kullanmak, aşırı vitellin membran, iç cam ring kenarından kaldırılır. Bakım pipet veya forseps ile membran delmek için alınır. </li><li> Embriyo hafifçe gevşek membranlar ve yumurta sarısı hücreleri temizlemek için tuzlu su ile durulanır. </li><li> 200 ul tuzlu su halkası (bu daha sonra transferi kolaylaştıracak) dış kenarına eklenir. </li><li> Montaj, ters bir plastik tabak ile kaplıdır. </li></ol> Bölüm 5: kültür inkübatör aktarılır <ol><li> Falcon kültürü çanak etiketlenir. </li><li> Ince albümin 2.5ml Falcon çanak altına eklenir. </li><li> Ince albümin 200μl Falcon çanak kapağın iç kenarına eklenir. </li><li> Ters çanak montaj kaldırılır. </li><li> Ince forseps kullanarak, cam halka izlemek cam kenarı boyunca slided ve Falcon çanak aktarılır. </li><li> Geri kalan tüm tuzlu su halka iç yüzeyine çıkarılır. </li><li> Falcon çanak kapağı kapalı ve mühürlü. </li><li> Montaj nemli odasında ve daha sonra inkübatöre yerleştirilir. </li><li> Embriyoların 38 ° C&#39;de 24 saat için kültür. </li></ol> Bölüm 6: Aşağıdaki kültürü, embriyo sabittir; kültür inkübatör aktarılır <ol><li> (Embriyolar in situ hibridizasyon için işlenmek üzere iseniz veya depc-PBS) fiksasyon çanak buz gibi soğuk PBS ile doludur. </li><li> Kültür inkübatör çıkartılıp hemen buz üzerinde yerleştirilir. Kültür çanak hemen buz gibi soğuk PBS / depc-PBS ile doludur. </li><li> Embriyo vitellin membran kopuk. Embriyo Pasteur pipeti küt ucunu kullanarak, fiksasyon çanak aktarılır. </li><li> Embriyo künt uçlu ince forseps kullanarak aşağı tutturulmuş. Fiksasyon çanak kapsayan PBS kaldırılır. Sabitleme eklendi [b] (notlara bakınız). </li><li> Uygulamaya bağlı olarak, embriyo 4 6-8 saat süreyle sabit ° C (Kriyostat), O / N 4 ° C (situs), ya da tüm montaj immünohistokimya RT az 1 saat. </li><li> Fiksatif buz gibi soğuk PBS / depc PBS ile kaldırılır ve değiştirilir. </li><li> In situ hibridizasyon veya immantibody boyama gibi alt uygulamalar için: sinir sistemi ve kalp, künt bir ucunu microcapillary iğne veya mikrodiseksiyon bıçak kullanarak delikli, bu daha sonraki adımda prob / antikor yakalama önleyecektir . Notlar: [a] Tuzlu oluşur: çözümü (1 lt): 121,0 g NaCl, 15.5 g KCl, 10.4 g CaCl 2 .2 H 2 O, 12,7 g MgCl 2 .6 H 2 O; B çözeltisi (1 litre) : 2.4 g Na 2 HPO 4 .2 H 2 O, 0.2 g NaH 2 PO 4 .2 H 2 O; Otoklav; Kullanmadan önce, 120 ml karıştırmak, 2700 ml H 2 O ile 180 ml ​​B. Mix [1] ; [b] Sabitleme (% 4 situs için PBS veya depc-PBS PFA) kullanımdan hemen önce hazırlanır. </li></ol> Bölüm 7: Temsilci Sonuçlar Embriyo kültür öncesinde, ilkel bir çizgi evre (HH4). Kültür dönemi sonunda, embriyo HH10 (2-3 mm uzunluğunda) geliştirilen ve kültür çanak merkezi görünür. Hücrelerden oluşan bir grup floresan DII carbocyanine etiket mümkündürSadece kültür önce (0h) ve kültür dönemi boyunca hareketini takip. Bu durumda, Hensen düğüm (HN) altındaki hücreler DII ile etiketlenir. Bu hücreler kademeli olarak gelişmekte olan Somitlerin (som) ve notokord (n) katkıda bulunmak için gösterilmiştir. <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" /> Bölüm 8: Sorun Giderme <table style="width: 698px; height: 241px;" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th> Sorun </th><th> Neden </th><th> Çare </th></tr><tr><td> Embriyo membran (adım 2) çıkıyorsa yerine sarısı ile kalır </td><td> Yumurta kalitesi zayıf </td><td> Mağaza yumurta aldıktan sonra 13 ° C; İstek taze yumurta üretilmektedir. Yumurta geliş tarihi aynı gün inkübe edin. </td></tr><tr><td> Vitellin membran slayt halka saatçilik cam aşağıdaki yerleştirme (3. adım) </td><td> Albumin kalıntıları </td><td> 2. adımda, tüm albümin eğimli forseps membran çekerek kaldırıldığını emin olun </td></tr><tr><td> Embriyo erişilemez: membran altında yatıyor (adım 4) </td><td> Membran yanlış tarafında yukarı doğru </td><td> 3. adımda, sarısı granülleri içeren membran tarafı yukarı bakacak şekilde emin olun. Parlak, cilalı yan aşağı bakmalıdır. </td></tr><tr><td> Embriyo (adım 6), tuzlu albümin / kültür sular altında </td><td> Tuzlu önce kültür halkasının içinde sol albümin /; zarı delik </td><td> 5. adımda, tuzlu / tüm albümin halkasının içinde kaldırılır olduğunu emin olun; 4. adımda, (künt sona erdi forseps kullanımı) forseps ile delmek membran yok emin olun </td></tr><tr><td> Embriyo aşağıdaki kültür parçalandı </td><td> Bakteriyel enfeksiyon </td><td> Antimikotik / antibiyotik tüm alet ve cam sterilize; kullanın. </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>

Yeni kültür yöntemi 2 büyüme faktörü tüm montaj boncuk 3, in situ hibridizasyon ve tüm montaj immünohistokimya 4 içeren greftleri arasında değişen çok çeşitli uygulamalar için de kullanılabilir. 24 saatlik süre içinde Kültür embriyonik gelişim zaman atlamalı hücre hareket analizi 5 veya GFP 6 electroporated yapıları içeren izleme gibi uygulamalar sürekli olarak izlenmesini sağlar.

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

Civciv embriyo erken embriyonik gelişim çalışmaya değerli bir araçtır. Şeffaflığı, erişilebilirliği ve kolay manipülasyon, bu oluşumu ve beyin, nöral tüp, hücre gruplarının ve kalp primordia desenlendirme eğitimi için ideal bir araçtır. Uygulamaları civciv embriyo kültürü, in situ hibridizasyon ve immünohistokimya, gen fonksiyonu, FGFs ve BMP gibi büyüme faktörü kaplı boncuklar greft yanı sıra tüm montaj analiz etmek için DNA veya RNA yapıları elektroporasyon içerir. Bu video civciv embriyo kültürü farklı adımları gösteriyor; İlk olarak, embriyo tuzlu su eksplante. Sonra, embriyo bir cam yüzük ortalanır. Embriyo çevreleyen zarların halka duvarları boyunca kaldırılır. Bu halka daha sonra, albümin bir havuzun bulunduğu bir kültür tabak içinde yer alır. 24 saat kadar embriyo kültür olduğu kültür çanak, kapalı ve nemli odasında yerleştirilir. Son olarak, embriyo, ring kaldırılır, sabit ve başka uygulamalar için işlenmiş. Bir sorun giderme kılavuzu da sunulmaktadır.

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

Bu video Yeni kültür, civciv embriyolarının 24 saat kadar yumurta dışında kültür olan bir yöntem gösteriyor. Bu yöntem, erken gelişme (14 som ilkel bir çizgi.) Fare E7-9 karşılık gelen bir süre çalışma sağlar. In situ hibridizasyon ve immünohistokimya, bu tekniğin Uygulamaları elektroporasyon içerir.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

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)

15.6K Görüntüleme.  Institute of Biosciences and Technology - Texas A&M Health Science Center. Bu video Yeni kültür, civciv embriyolarının 24 saat kadar yumurta dışında kültür olan bir yöntem gösteriyor. Bu yöntem, erken gelişme (14 som ilkel bir çizgi.) Fare E7-9 karşılık gelen bir süre çalışma sağlar. In situ hibridizasyon ve immünohistokimya, bu tekniğin Uygulamaları elektroporasyon içerir.

Video: 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.

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

Biyoloji

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.

Civciv Ex ovo Kültür ve Ex ovo CAM Testi: Gerçekten Nasıl Çalışır?

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.

Organotipik murin Retina Wholemount Kültür in vivo

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.

Rotator-tipi Şişe Kültür Sistemi kullanılarak Kemirgen Tüm Embriyo Kültürü Yöntemi

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.

Ex Vivo Kültür İlköğretim İnsan Fallop Tüp Epitel Hücreleri

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.

Erken Civciv Embriyo morfogenetik Doku deformasyonlar Takibi

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.

Yeni çok katmanlı bir balon kullanarak Memeli Hücre Kültürü Ölçeği-Up

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.

Yeni Bir<em&gt; Ex vivo</emEmbriyonik Fare Kalp için&gt; Kültür Yöntemi

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.

Eksplantasyon Kültüründe Diş mikropların Geliştirme takiben için Dilim Kültür Yöntemi

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.

Fare Embriyonik Cilt ve Melanoblast Göç Canlı görüntüleme ex vivo Kültür

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

Methods Article

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi