이 작품은 Saban 연구소 사전 박사 수상 (PMDM)에서 지원하고, NIH RO1 HL75773 (DW)에 의해.했습니다

오르간 문화는 유전자와 단백질 발현을 연구하기 위해 매우 유용하고 다재 다능한 기술입니다. 이 전 생체내 문화 방법은 사전 또는 생체내 연구에 보완에 사용할 수 있습니다. 1. 배아 룽 절연 <ol><li> 시간 초과 - 임신 생쥐는 NIH와 OLAW 지침에 따라 CO 2 마취법에 의해 postcoitum 일 11.5 또는 12.5 (E11.5 - 12.5)에 희생됩니다. 동물은 챔버에 배치되며 100 % CO 2가 도입됩니다. 동물이 의식이 후 CO 2 흐름이 증가됩니다. 동물의 임상 죽음을 보장해야합니다. </li><li> 모든 다음 단계는 층류 후드에서 무균 조건 하에서 완료해야합니다. 자궁은 임신 여성에서 제거됩니다. 동물의 자궁을 제거하려면 임신 여성 자신의 뒤쪽에 위치하고 70 % 에탄올로 뿌렸 수 있습니다. 절개는 복부에 만들어이며, 피부는 동물의 뒷다리를 누른 상태에서 위쪽으로 피부를 당겨 제거됩니다. 복막 캐비티가 열립니다과 자궁은 동물에서 제거됩니다. </li><li> 혈액은 추운 행크스 밸런스드 소금 솔루션 (HBSS)으로 가득 50 ML 원뿔 관에 자궁을 배치 해제 씻어서이며, 튜브가 부드럽게 2 분 누볐어입니다. </li><li> 자궁은 다음 입체 해부 현미경 페트리 접시에 배치하고, 배아는 봄 가위를 사용하여 자궁 벽을 절개하여 자궁에서 발표됩니다. 배아는 천공 숟가락을 사용하여 수확하고, HBSS를 포함하는 새로운 배양 접시에 얼음에 표시됩니다. </li><li> 입체 해부 현미경, 배아 폐가 차가운 HBSS (그림 1)이있는 페트리 접시에 하나씩를 해부하고 있습니다. </li><li> 분리된 배아 허파는 살균 전송 피펫을 사용하여 HBSS를 포함하는 페트리 접시에 얼음에 위치합니다. </li></ol> 2. 배아 룽 문화 <ol><li> 1 밀리리터에 따라 잘 D-MEM/F-12 500 microliters의 페니실린 - 스트렙토 마이신의 50 단위 / ML과 보충은 Nunclon의 폴리스티렌 접시에 추가됩니다. </li><li> Nuclepore 폴리 카보 네이트 트랙 엣지 분리막은 미디어의 상단에 위치합니다. Nuclepore 폴리 카보 네이트 트랙 엣지 분리막은 미디어의 상단에 배치되면, 빛나는 측면은 미디어, 그리고 거친 면을 위쪽에 직면에 대해되어 있는지 확인합니다. </li><li> 해부 배아 폐암은 살균 전송 피펫을 사용하여 Nuclepore 폴리 카보 네이트 트랙 엣지 분리막 위에 위치합니다. </li><li> 배아 폐 위치는 포셉를 사용하여 조정됩니다. 필터에 위치 배아 허파가 손상 기관이 있어야하고 바로 입장을 가지고 그들의기도와 배치, 따라서 모든 폐 같은 내부 압력을 가지고 있도록합니다. </li><li> Nunclon의 폴리스티렌 요리는 세포 배양기 (그림 2)에서 원하는 문화 시간에 전송됩니다. </li></ol> 3. 자료 1. 배아 전체 룽 격리 <ol><li> 시간이 초과되었습니다 - 임신 (C57BL6 야생 입력하거나 유전자 변형) 생쥐 13.5 (E11.5 - 13.5)에 postcoitum 일 11.5에 희생 될 수 있습니다. </li><li> 염화칼슘, 염화 마그네슘 또는 마그네슘 황산없이 행크스 &#39;밸런스드 소금 솔루션 (HBSS) (1X), 액체,. (Invitrogen, 칼스 배드, CA) 50 단위 / 페니실린 - 스트렙토 마이신의 ML (Invitrogen, 칼스 배드, CA)과 보완. 실온에서 4 ° C에서 개통 후 HBSS를 저장합니다. 나누어지는하고 보관 -20 ° C.에 페니실린 - 스트렙토 마이신 </li><li> 입체 해부 현미경 (Leica, Wetzlar, 독일). </li><li> 더몬트 포셉, 파인 이리스 가위 (직선), 노예스 봄 가위와 모리아 ® 스푼 (천공) (파인 과학 도구, 포스터 시티, CA)를 포함 해부로드 공구를 추천하십시오. </li></ol> 2. 배아 전체 룽 문화 <ol><li> 중간 Dulbecco의 수정된 이글 : 영양소 믹스 F - 12 (D-MEM/F-12) (1X), 액체, 1:1 L - 글루타민을 포함하지,하지만 HEPES 버퍼. (Invitrogen, 칼스 배드, CA) 50 단위 / 페니실린 - 스트렙토 마이신의 ML (Invitrogen, 칼스 배드, CA)과 보완. 4 DMEM/F-12 거리 조명 (예 : 알루미늄 호일에 포함)의 병 및 저장 ° C. 유지 나누어지는하고 보관 -20 ° C.에 페니실린 - 스트렙토 마이신 </li><li> Nuclepore 폴리 카보 네이트 트랙 엣지 분리막, 8.0 - M, 13mm (왓먼 Nuclepore 트랙 엣지 멤브레인 (왓먼, 플로럼 공원, NJ). </li><li> 뚜껑과 Nunclon의 폴리스티렌 요리, 4 우물 (로체스터, NY). </li><li> 일회용 전송 pipettes, 멸균 (피셔 과학, 피츠버그, PA). </li></ol><img src="/files/ftp_upload/2035/2035fig1.jpg" alt="그림 1" /> 그림 1. E12 배아 폐의 해부. (A) E12.5 전체 난자는 오른쪽에서 볼. (B)를 마우스 오른쪽 forelimb이 배아에서 제거되었습니다. (C) 집게는 접시에 안정적인 배아를 들고 왼손으로 개최. 화살표가 (D) 배아의 폐 마음 (제거하기 위해 사후 거짓말을 해부의 계획을 나타냅니다척추)과 앞쪽에. 이 그림은 피부와 심장 제거 후 폐를 보여줍니다. (E) 인두 제거는 배아의 손상 폐 분리 수 있습니다. (F) 외부 pharyngeal 조직과 식도가 떨어진 손질했습니다. 그대로 기관과 후두와 해부 E12.5 배아 폐암이 표시됩니다. (CR) 두개골 엽 (클럽메드) 중간 엽 (CA) 꼬리의 엽 (ACC) 액세서리 엽 (르) 왼쪽 엽. <img src="/files/ftp_upload/2035/2035fig2.jpg" alt="그림 2" /> 그림 2. FGF9은 mesenchyme의 확장과 체외에서 성장 폐암에서 상피의 팽창을 유도. (AC) E12.5 폐가 FGF9의 부재에서 48 시간 동안 성장합니다. 분기의 증가에 유의하십시오. (DF) E12.5 폐는 FGF9의 존재에 48 시간 동안 성장. 문화 24 시간 후 빠르면 상피와 mesenchyme (E)의 팽창을 확인합니다. 48시간 (F) 후, 상피에 대한 효과는 더욱 발음입니다. 스케일 바 : AF 400 μm의 4. 이 실험 방법에 대한 전체 텍스트 프로토콜에서 사용할 수 있습니다 <a href="http://springerprotocols.com/Abstract/doi/10.1007/978-1-59745-019-5_5">스프링거 프로토콜</a> .

<ol>
	<li>Alescio, T., Cassini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+in+vitro+of+tracheal+buds+by+pulmonary+mesenchyme+grafted+on+tracheal+epithelium.">Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium.</a> J Exp Zool. 150, 83-94 (1962).</li><li>Warburton, D., Schwarz, M., Tefft, D., Flores-Delgado, G., Anderson, K. D., Cardoso, W. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+lung+morphogenesis.">The molecular basis of lung morphogenesis.</a> Mech Dev. 92, 55-81 (2000).</li><li>Spooner, B. S., Hardman, P., Paulsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gravity+in+mammalian+organ+development:+differentiation+of+cultured+lung+and+pancreas+rudiments+during+spaceflight.">Gravity in mammalian organ development: differentiation of cultured lung and pancreas rudiments during spaceflight.</a> J Exp Zool. 269, 212-222 (1994).</li><li>Moral, d. e. l., M, P., De Langhe, S. P., Sala, F. G., Veltmaat, J. M., Tefft, D., Wang, K., Warburton, D., Bellusci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+FGF9+on+epithelium+and+mesenchyme+in+mouse+embryonic+lung.">Differential role of FGF9 on epithelium and mesenchyme in mouse embryonic lung.</a> Dev Biol. 293, 77-89 (2006).</li><li>Moral, D. e. l., M, P., Sala, F. G., Tefft, D., Shi, W., Keshet, E., Bellusci, S., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-A+signaling+through+Flk-1+is+a+critical+facilitator+of+early+embryonic+lung+epithelial+to+endothelial+crosstalk+and+branching+morphogenesis.">VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis.</a> Dev Biol. 290, 177-188 (2006).</li><li>Jaskoll, T. F., Don-Wheeler, G., Johnson, R., Slavkin, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+mouse+lung+morphogenesis+and+type+II+cytodifferentiation+in+serumless,+chemically+defined+medium+using+prolonged+in+vitro+cultures.">Embryonic mouse lung morphogenesis and type II cytodifferentiation in serumless, chemically defined medium using prolonged in vitro cultures.</a> Cell Differ. 24, 105-117 (1988).</li><li>Seth, R., Shum, L., Wu, F., Wuenschell, C., Hall, F. L., Slavkin, H. C., Warburton, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+epidermal+growth+factor+expression+in+early+mouse+embryo+lung+branching+morphogenesis+in+culture:+antisense+oligodeoxynucleotide+inhibitory+strategy.">Role of epidermal growth factor expression in early mouse embryo lung branching morphogenesis in culture: antisense oligodeoxynucleotide inhibitory strategy.</a> Dev Biol. 158, 555-559 (1993).</li></ol>

관심 없음 충돌 선언하지 않습니다.

mouse embryonic lung culture, a system to evaluate the molecular mechanisms of branching

폐 원시 사양뿐만 아니라 분기 morphogenesis, 다양한 폐 세포 lineages의 형성은 주변 mesenchyme과 mesothelium과 폐 endoderm의 특정 상호 작용을 필요로합니다. 폐 mesenchyme는 폐 분기 morphogenesis을위한 유도 신호의 소스로 표시되었습니다. 상피 - mesenchymal - mesothelial 상호 작용은 또한 배아 폐 morphogenesis에 중요합니다. 초기 배아 폐 기관의 문화는 상피 - mesenchymal 상호 작용을 연구하기 위해 매우 유용한 시스템입니다. 쉽게 (산모 영향과 혈액 흐름의 부재에서)이 시스템에서 조작할 수있는 구체적인 조건 하에서 상피와 mesenchymal 모두 morphogenesis 진행. 더 중요한 것은이 기술은 쉽게 serumless, 화학적으로 정의 문화 미디어로 이루어 질 수 있습니다. 함수의 이득과 손실 표현 단백질, 재조합 바이러스 벡터 및 / 또는 유전자 변형 마우스 변종, 안티 센스 RNA뿐만 아니라 RNA 간섭 유전자 최저 분석을 사용하여 얻을 수 있습니다.

Watch this Scientific Journal Video about Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching at JoVE.com

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

초기 배아 폐 기관의 문화는 상피 - mesenchymal 상호 작용을 연구하기 위해 매우 유용한 시스템입니다. 쉽게이 시스템에서 조작할 수있는 구체적인 조건 하에서 상피와 mesenchymal 모두 morphogenesis 진행.

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 ...

mouse-embryonic-lung-culture-system-to-evaluate-molecular-mechanisms

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18.3K 조회수.  Childrens Hospital Los Angeles. 초기 배아 폐 기관의 문화는 상피 - mesenchymal 상호 작용을 연구하기 위해 매우 유용한 시스템입니다. 쉽게이 시스템에서 조작할 수있는 구체적인 조건 하에서 상피와 mesenchymal 모두 morphogenesis 진행.

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

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

배아 마우스 신장의 Nephrogenic 지대의 세포의 분리 및 문화

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

배아 림브 스킨 Vasculature에 대한 전체 마운트 Immunohistochemical 분석 : 엠브료에 혈관 분기 Morphogenesis를 공부하는 모델 시스템

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

에 Postembryonic Phenotypes을 식별하기 위해 RNAi 심사 C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Blastomere Explants는 배아 개발하는 동안 세포 운명의 헌신에 대해 테스트 할

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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 ...

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 ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

절차 식물 추출물에서 분산 된 입자의 제거를위한 응집제의 효율성을 평가하는

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

점액 아가로 오스 겔 전기 영동 : 높은 분자량 당 단백질에 대한 웨스턴 블로 팅


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...