우리는 중요한 원고의 읽기 및 자금 지원을위한 NIH (부여 # R01HL061656)의 안드레아 포트 베리에게 감사의 말씀을 전합니다.

1. 배아의 마음을 절개 <ol><li> 승인 안락사 기술을 사용하여 원하는 배아 일에 시간 초과 임신 마우스를 안락사. 모든 실험은 채플 힐에서 노스 캐롤라이나 대학에서 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다. </li><li> 대충 해부하기 전에 70 % 에탄올로 여성 스프레이. 자궁을 검색하고 소비세하는 여성의 복강을 엽니 다. </li><li> 차가운 1X 인산염 완충 식염수 (PBS)와 필요에 린스를 포함하는 페트리 접시에 자궁을 놓습니다. 첨부 된 배아 주머니를 노출 정중선을 통해 자궁을 열고 잘라. </li><li> 배아 주머니를 열고 잘라 배아를 검색 할 수 있습니다. 차가운 PBS로 두 번째 페트리 접시에 배아를 놓습니다. 얼음 자궁 경적 페트리 접시를 돌려줍니다. </li><li> 가슴 벽에 대한 액세스를 개선하기 위해 태아의 목을 베십시오. 유전자형이 필요한 경우, 제거하고 얼음에 배치 에펜 도르프 튜브에 꼬리를 저장합니다. </li><li> 와그것의 뒤에 태아는 심장과 폐를 시각화하는 가슴 벽을 열고 잘라. 단계에 따라 가슴 벽이 투명 할 수 있습니다. </li><li> 조심스럽게 집게를 사용하여 마음을 들어 올려 아래의 혈관을 잘라. 그런 마음을 해방하는 큰 그릇 위에 잘라. 필요한 경우, 불필요한 조직을 제거합니다. </li><li> 차가운 PBS로 가득 찬 24 잘 접시의 잘 하나에 마음을 놓고 얼음에 접시를 놓습니다. </li><li> 마음은 모든 배아에서 적출 될 때까지 이전 단계를 반복합니다. </li></ol> 2. 문화 셋업 <ol><li> 층류 후드에 1:1 비율로 차가운 리겔하고 원하는 문화 매체 (Dulbecco의 수정 이글 중간 (DMEM) 예를 들면 10 % 태아 소 혈청 (FBS)) 결합. 미리 따뜻한 문화 매체하지 않습니다. </li><li> 얼음도 얼음에 유지되는 24 잘 배양 접시의 잘으로 희석 용액의 피펫 500 ~ 1,000 μL의 희석 마트 리젤을 유지. </li><li> 후드에서 신중하게 픽업과 절제를 배치마트 리젤 함유 배양 접시의 잘 심장 얼음에 문화 접시를 유지하면서. </li><li> 임베디드 마음의 방향이 매우 중요합니다 경우 대충 70 % 에탄올로 거꾸로 현미경과 주변 공간을 스프레이. 후드 반면, 여전히 얼음 마트 리젤 함유 문화 판의 잘 절제의 마음을 놓으십시오. 그런 다음, 현미경 플레이트를 배치하고 신속하게 방향을 절제 마음을 마트 리젤이 응고하기 시작. </li><li> 가습 37 ° C 배양기 (5 % CO 2)에 배양 접시를 놓고 마트 리젤 약 30 분 동안 응고 할 수 있습니다. </li><li> 잘 마음을 포함하는 각에 1 ML 미리 예열 문화 매체를 추가합니다. </li><li> 마음은 적어도 나흘 문화에 남아있을 수 있습니다. 배지 2 일마다 변경하는 것이 좋습니다. </li></ol> 3. 고정 및 시각화 <ol><li> 원하는 경우, 살아있는 마음을 시각화하기 위해 (예를 들어 SYTO-16) 형광 라이브 세포 염료를 추가합니다. 사진이 될 것입니다이 포함되었을 때의 위치에 따라 볼 수있는 마음의 측면에 제한됩니다. </li><li> 후 배양 분석 (예 : 전체 마운트 영상 또는 단면)을 수행 할 경우, 배양액을 제거하고 차가운 4 % 포름 알데히드로 교체합니다. 30 분 동안 얼음에 접시를 놓습니다. </li><li> 차가운 포름 알데히드와 얼음 마트 리젤의 용해를 촉진 한 후, 신선한 포름 알데히드와 (과 마트 리젤) 정착액을 교체하고 4에서 하룻밤 마음 ° C.를 해결 </li><li> 4 ° C까지 추가 분석에서 버퍼 (예 : PBS 또는 트리스) 및 저장과 마음을 씻으십시오. </li></ol>

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저자는 그들이 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

현재 문화권 시스템은 마우스 배아 심장 연구에 상당한 이점을 포즈. 이 문화 시스템도 문화 사일 후 괴사의 제한 징후와 심근 수축력과 관상 동맥 신경총을 보존합니다. 또한, 반 고체 매트릭스 문화 동안 자리에 코팅 구슬을 보유하는 것이 계약의 유연성을 허용하면서 개발 중심의 3 차원 형태를 유지하기 위해 충분한 지원합니다. 이러한 지원에도 불구하고,이 행렬은 살아있는 마음의 형광 분?...

최근 몇 년 동안, 형질 전환 마우스의 개발 심장 결함을 공부 지배적 인 모델 시스템이다. 그러나, 제브라 피쉬와 같은 다른 모델 생물은 마우스에 비해 상당한 장점을 가지고 입증했다. 제브라 피쉬의 세 가지 주요 장점은 배아에 대한 접근의 용이성을 위해, 계란의 외부 누워있다, 심장 개발을 쉽게 시각화 할 수 배아의 광학 투명성, 그리고에 작은 분자 치료를 적용의 용이성은 배아의 발달을 조절 1. 따라서, 배아 기관의 전직 자궁 내 성장을 허용하는 문화 기술의 발전은 적어도 부분적으로 제한 현재 형질 전환 생쥐의 발달 과정을 공부하는 연구자에 의해 경험 우회 것이다. 생체 심장 배양 시스템은 DIF 방법 작은 분자 및 분석 치료를 허용하는 병아리와 마우스 배아 모두에서 개발 된마음의 ferent 지역은 2-6 통신합니다. 전체 마우스 심장 문화, 마음 배아로 배아에서 채택 (E) 연령 12.5 흔들 2,3,5의 유무에 관계없이 배지에 배치 할 수 있습니다. 이 기술을 사용하여, 배아 마음은 성공적 E13.5의 동등성을 배양하고 있고, 흔들 배양 마음은 오랫동안 3 일 (5 E10.5에서 시작) 3로 유지되고있다. 그러나, 연구는 이전 배아에서 마음의 성공적인 문화를보고있다. 마찬가지로, 구조 실험은 배양 배지 2 전 세계적으로 치료제를 적용 제한되었습니다. 마음은 절제 내장하고 vibratome를 사용하여 절편되는 슬라이스 문화 시스템은, 또한 E12.5 마우스 마음과​​ 햄버거 - 해밀턴 단계 36 (약 E16 마우스) 병아리 마음으로 두 어린 마음에 활용 된 이러한 출생 후 성인 마우스를 H로 2,4,6, 세 마음,earts 성인 인간의 마음 7,8. 배아 분석은 일반적으로 150 μm의 두께 섹션 2.4을 활용하고 있지만, 단면의 두께는 산소 부족 8 증거도없이 큰 500로 μm의 수 있습니다. 이러한 슬라이스 문화는 대부분 조각이 기간 9 내내 수축성 유지 보수와 문화 2 개월만큼 유지되고있다. 고립 된 심근의 연구에 비해, 이러한 슬라이스 문화는 이웃 세포 ​​유형과 심근의 공동 문화를 허용하고 생체 분석을위한 유용한 방법을 제공합니다. 그러나, 이러한 문화는 단순히 문화 매체의 마음을 (예 : vibratome에서 단면의 살아있는 심장을 포함) 배치보다 더 정교한 셋업이 필요하며 모든 분석은 분명 섹션에서 마음의 부분으로 제한됩니다. 제한 배양 배아 마우스 마음과​​ 형질 전환 생쥐 AVA의 재산에 대해 위에서 설명한 주어연구 ilable, 우리는 위버에 의해 개발 된 생체 폐 배양 시스템과 유사한 생체 마우스 심장 배양 시스템을 개발 하였다. 10 우리의 문화 시스템은 전체 배아 마우스 마음에서 리모델링 관상 동맥 순환의 장기 문화와 시각화를 허용합니다. 또한, 마트 리젤의 사용에 따라서 치료 에이전트와 지역화 된 치료를 제공, 구슬이 심장에 가까운 장소에서 개최 할 수 있습니다. 이 실험은 관상 동맥 형성 등의 과정에서 주어진 치료의 효과를 비교하기 위해 다른 개발 시점에서 수행 할 수 있습니다. 작은 분자는 리겔을 통해 확산 할 수 있기 때문에,이 문화 시스템은 또한 특정 세포 - 세포 연락처가 특정 개발 프로세스에 필요하거나 다른 한 지역에서 paracrine 신호가 있는지 여부를 확인하는 서로 가까이 심장의 문화를 해부 지역에 사용할 수 있습니다 필요. 이 배양 시스템비교적 간단하고, 슬라이스 문화 시스템과는 달리, 기본적인 문화 시약 및 대부분의 실험실에서 쉽게 사용할 수있는 셋업을 사용합니다. 간단히, 절개 배아의 마음은 반 고체 지원을 제공 희석 리겔에 배양한다. 이 지원은 계약의 마음을 허용하면서 심장의 3 차원 형태를 유지하기에 충분합니다. 이 시스템을 사용하여, 이전 마우스 배아 (E14.5-E16.5)의 전체 마음은 최대 네 개의 일을위한 문화 유지 될 수 있습니다. 전체 관상 동맥 혈관 따위는 슬라이스 문화 달리, 유지, 그래서 마음의 다른 지역에서 발생하는 신호 단서가 존재 남아있다. 또한, 살아있는 세포 형광 염료는 살아있는 마음의 시각화를 허용하도록 마트 리젤을 침투 할 수 있고, 단백질 복합 구슬 지역화 된 신호 소스를 제공하기 위해 심장 근처에 배치 할 수 있습니다. 함께, 이러한 장점은이 기술에게 embryon의 발달 과정을 연구하기위한 이상적인 방법을IC 마우스 마음입니다.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Timed-pregnant mice</td>
 <td></td>
 <td></td>
 <td>To be dissected at the embryonic stage of interest</td>
 </tr>
 <tr>
 <td>PBS (1x)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Cellgro </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Sigma-Aldrich </td>
 <td>F2442 </td>
 <td></td>
 </tr>
 <tr>
 <td>Growth factor-reduced Matrigel</td>
 <td>BD Bioscience </td>
 <td>356231 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syto-16</td>
 <td>Invitrogen </td>
 <td>S7578 </td>
 <td>Used as directed in 13 </td>
 </tr>
 <tr>
 <td>24-well culture plate</td>
 <td>Fisher Scientific</td>
 <td>07-200-84</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Stereoscopic microscope</td>
 <td>Nikon </td>
 <td>SMZ645</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture incubator</td>
 <td>Thermo </td>
 <td>3110 </td>
 <td></td>
 </tr>
</tbody>
 </table>

a novel ex vivo culture method for the embryonic mouse heart

마우스 발달 연구는 임신 기간 동안 태아의 어려움에 의해 방해된다. 따라서, 프로토콜은 분리하고 그 문화를 개별 기관이 모두 발달의 변화를 시각화하고 있도록 새로운 치료 전략의 방법을 제공하는 것이 필수적이다. 임신의 후반 단계에서 배아 심장의 장기 문화를 촉진하기 위해, 우리는 절제 마음이 반 고체, 희석 리겔에 배양되는 프로토콜을 개발했다. 이 기판은 3 차원 구조를 유지하기 위해 충분한 지원 제공하지만 지속적인 수축을 허용 할만큼 충분히 유연합니다. 간단히 말해서 마음이 배아에서 절제와 추위 마트 리젤의 혼합물에 배치 성장 매체로 1:1 희석. 희석 마트 리젤이 굳은 후 성장 배지 배양 접시에 추가됩니다. 배아 일 16.5로 늦게 절제의 마음 나흘 후 해부 가능한 하였다. 관상 동맥 신경 얼기의 분석이 방법은하지 않는 것을 보여줍니다관상 동맥 혈관 발달을 방해. 따라서, 우리는 배아 마음의 장기적인 문화의 새로운 방법을 제시한다.

이 기술을 사용하여, 마음은 3 차원의 형태를 유지하고 가능한 유지 등의 지속적인 수축 (동영상 1)로 표시. 이러한 수축은 심실에 비해 심방에서 지속적으로 더 유명하다. 문화에 따라, 마음은 고정 및 처리 특정 마커 식 또는 구조를 검사하는 면역 또는 조직학도합니다. 그림 1A는 고정, 24 시간 동안 배양 한 배아 마우스 심장의 심실과 대 혈관의 기초를 보여줍니다 대해 ?...

Watch this Scientific Journal Video about A Novel Ex vivo Culture Method for the Embryonic Mouse Heart at JoVE.com

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

마우스 발달 연구는 임신 기간 동안 태아의 어려움에 의해 방해된다. 임신의 후반 단계에서 배아 심장의 장기 문화를 촉진하기 위해, 우리는 절제 마음이 반 고체, 희석 리겔에 배양되는 프로토콜을 개발했다.

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 ...

a-novel-ex-vivo-culture-method-for-the-embryonic-mouse-heart

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JoVE Journal

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

13.2K 조회수.  University of North Carolina at Chapel Hill. 마우스 발달 연구는 임신 기간 동안 태아의 어려움에 의해 방해된다. 임신의 후반 단계에서 배아 심장의 장기 문화를 촉진하기 위해, 우리는 절제 마음이 반 고체, 희석 리겔에 배양되는 프로토콜을 개발했다.

비디오: A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

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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생물학

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

마우스 망막 신경절 세포의 유전자 발현에 대한 utero 및 전직 생체내의 Electroporation에

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

마우스 배아의 폐 문화, 분기의 분자 메커니즘을 평가하는 시스템

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

인간의 장내 점막의 Explants의 문화와 편광 자극을위한 새로운 방법

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

배아 심실 내피 세포의 분리

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

무 혈청 전체 배아 배양 시스템에서 마우스 배아 개발

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

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 ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

개발 배아 마우스 심장 밸브 지역에서 단백질 분리

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

은 C-FOS 단백질 면역 조직 검색 : 특정 생리적 반응에 관여 중앙 진학의 마커로서 유용한 도구<em&gt; 생체</em&gt; 및<em&gt; 전의 VIVO</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...