우리는 산소 챔버를 제공하는 우수한 기술 지원 및 박사 안토니오 디 Sabatino의를 위해 에리카 Mileti 감사합니다. 자금 조달 : MR에와 폰다 치오 Cariplo와 마리 퀴리 국제 교육 이동성 네트워크 (크로스 토크 부여 계약 번호 : 21553-2)에 의해 지원 :이 작품은 7 EU 프레임 워크 프로그램 (Dendroworld IBDase, ERC)에서 교부금에 의해 지원되었다 KT.

1. 얻기 및 조직 준비 <ol><li> 조직 (건강 또는 IBD 점막)이 수술하는 동안 얻을 수 있습니다. 일단 시편은 4 ° C에서 또는 얼음에 펜 / Strep의로 보충 칼슘 + + / 마그네슘 + + 무료 HBSS 버퍼에 유지 가능한 빨리 실험실로 전송을 절제합니다. </li></ol> 시편의 * 사이즈는 조직의 가용성에 따라 달라 얻은 조직의 부분은, 진단을 위해 필요하지 않습니다 것을 엄격 건강하고 IBD 주제 사이에서 크게 다를 수 있습니다. 건강한 조직은 대장 암 수술을받은 환자에서 절제합니다. <ol start="2"><li> 부드럽게 조직을 세척하고 모든 장간막 물질을 청소합니다. 페트리 접시 (반드시 immerged되지 않음)에 HBSS 버퍼에 조직을 유지하면서 멸균 가위와 집게를 사용하여 점막하에서 점막 층을 분리합니다. 가능한 조금 집게와 점막 표면을 터치합니다. </li><li> 최소 1 C는 줄무늬의 점막을 잘라m 폭과 가능한 한 오래. 당신의 음식을 절단하는 것처럼 많은 두 멸균 메스를 사용합니다. </li><li> HBSS에 부드럽게 점막을 청소하고 위쪽을 향하게 정점면 페트리 접시에 그것을 확장 씻으십시오. </li></ol> 2. 조직을 설치 <ol><li> 새로운 배지 (tisDMEM)를 준비합니다. DMEM는 글루타민 (2 ㎜)와 NaPyr (1 ㎜)로 보완 사용하고 FBS - 나 15 %를 추가 (태아 혈청 - 북미), 1 % ITS-X 및 사용시 200 NG / ML EGF. </li><li> FBS - 나 (금속 그리드가 완전히 빠져들 수 있도록 약 30 mL를 사용한다) 및 잠수함 금속 격자의 나머지 부분과 10cm 페트리 접시를 채우십시오. 접시는 꼭지 플라스틱 실린더를 열고 지원하십시오. </li></ol> * 금속 격자 철의 만들어집니다. 메쉬 0.5 mm의 측면 사각형 모양입니다. <ol start="3"><li> 10 20 μL 피펫 수술 접착제의 3 μL를 가져 가라. 이 f를 누른 상태에서 플라스틱 실린더의 경계 중 하나에 조심스럽게 적용집게와 IRM. </li><li> 부드럽게 최대한 가까이 스트립의 경계 중 하나, 점막의 정점 측에 실린더를 놓습니다. </li><li> 물론 중앙에 매체의 850 μL-1 ML을 분배하고 플레이트의 중앙 우물의 가장자리에 금속 격자를 지원 단단히 조직에서 실린더를 연결하는 접착제 시간을주고, 가운데 잘 장기 배양 플레이트를 준비합니다. </li><li> 멀리 이전과 같이 두 개의 멸균 메스를 사용하여 실린더의 경계에서 여분의 조직을 잘라. </li><li> 부드럽게 연결된 조직으로 실린더를 들고 접시의 중앙에 금속 격자의 기저 측면을 놓습니다. </li></ol> 3. 자극 <ol><li> 원하는 농도 선택의 자극을 사용합니다. </li><li> 피펫 팁 실린더 또는 점막 표면을 방해하지 않고 플라스틱 실린더의 중앙에 적절한 볼륨을 적용합니다. 선택한 볼륨을 균일하게 실린더 내부 표면을 커버해야합니다. 에dicated 볼륨 : 큰 플라스틱 실린더 : 200 μL 보통 플라스틱 실린더 : 100 μL 작은 플라스틱 실린더 : 50 μL </li><li> 까지 기존의 배양기에서 3 시간에 대한 품어. </li></ol> * 당신은 긴 시간 포인트를 배양하고자하는 경우 (3.5 참조) 자극의 아주 작은 볼륨 (20 μL)을 사용하여 확인하고 직접 1 기압의 압력에서 100 % O 2 분위기에서 조직을 배치 . <ol start="4"><li> 혀끝의 얼굴에 매체의 두꺼운 층이 가스 교환 수없는 경우 조직이 2 O 충분하지 않기 때문에, 조직의 혀끝의 표면에 떨어져 자극으로 매체를 가지고 신선한 배지로 교환하지 않는다. 플레이트를 커버. </li><li> 1 기압의 압력에서 100 % O 2 분위기에서 조직을 놓습니다. </li></ol> 4. 수확 <ol><li> 전체 문화 시간 (± 2.5 시간) 24 시간 후주의 일에서 실린더를 제거전자 메스 부드럽게 긁어 접착제 오프의 도움으로 조직하고 (RNA 정화 및 유전자 발현 프로파일 또는 단백질 정제와 서양 얼룩 분석 액체 N2의 면역 및 / 또는 면역 분석, 또는 스냅 동결 수정하여 원하는 분석에 따라 저장 ). </li><li> 사이토 카인 분비 프로파일에 대한 기저 매체를 수확. 펠렛 파편에 7 분 동안 8,000 rpm으로 원심 분리, 에펜 도르프 튜브 (또는 분취)의 상층 액을 전송하고 즉시 -20 ° C.에 저장 </li></ol> * 당신이 이상 상층 액을 유지하려는 경우, -80에서 보관 ° C.를

<ol>
	<li>Schuller, S., Lucas, M., Kaper, J. B., Giron, J. A., Phillips, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ex+vivo+response+of+human+intestinal+mucosa+to+enteropathogenic+Escherichia+coli+infection.">The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.</a> Cell. Microbiol. 11, 521-530 (2009).</li><li>Tsilingiri, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+and+postbiotic+activity+in+health+and+disease:+comparison+on+a+novel+polarised+ex-vivo+organ+culture+model.">Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model.</a> Gut. , (2012).</li><li>Tsilingiri, K., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Should+probiotics+be+tested+on+ex+vivo+organ+culture+models?.">Should probiotics be tested on ex vivo organ culture models?.</a> Gut Microbes. , (2012).</li><li>Mileti, E., Matteoli, G., Iliev, I. D., Rescigno, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+immunomodulatory+properties+of+three+probiotic+strains+of+Lactobacilli+using+complex+culture+systems:+prediction+for+in+vivo+efficacy.">Comparison of the immunomodulatory properties of three probiotic strains of Lactobacilli using complex culture systems: prediction for in vivo efficacy.</a> PLoS One. 4, e7056 (2009).</li><li>Cencic, A., Langerholc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+cell+models+of+the+gut+and+their+applications+in+food+microbiology--a+review.">Functional cell models of the gut and their applications in food microbiology--a review.</a> Int. J. Food Microbiol. 141, S4-S14 (2010).</li><li>te Velde, A. A., Verstege, M. A., Hommes, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+appraisal+of+the+current+practice+in+murine+TNBS-induced+colitis.">Critical appraisal of the current practice in murine TNBS-induced colitis.</a> Inflamm. Bowel Dis. 12, 995-999 (2006).</li><li>Senior, P. V., Pritchett, C. J., Sunter, J. P., Appleton, D. R., Watson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+regeneration+in+adult+human+colonic+mucosa+during+prolonged+organ+culture.">Crypt regeneration in adult human colonic mucosa during prolonged organ culture.</a> J. Anat. 134, 459-469 (1982).</li><li>Browning, T. H., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+culture+of+mucosal+biopsies+of+human+small+intestine.">Organ culture of mucosal biopsies of human small intestine.</a> J. Clin. Invest. 48, 1423-1432 (1969).</li><li>Besselink, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotic+prophylaxis+in+predicted+severe+acute+pancreatitis:+a+randomised,+double-blind,+placebo-controlled+trial.">Probiotic prophylaxis in predicted severe acute pancreatitis: a randomised, double-blind, placebo-controlled trial.</a> Lancet. 371, 651-659 (2008).</li><li>Lakhdari, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+NF-kappaB+modulation+capabilities+within+human+intestinal+commensal+bacteria.">Identification of NF-kappaB modulation capabilities within human intestinal commensal bacteria.</a> J. Biomed. Biotechnol. 2011, 282356 (2011).</li></ol>

관심 없음 충돌 선언하지 않습니다.

인간의 장 점막에 대한 치료가 유효하게 테스트 할 수있는 생리학 관련 모델에 대한 필요성은 오랫동안 강조하고있다. 많은 연구자들은 그 목적을 위해 지금까지 사용한 셀 5와 마우스 모델 모두 6 잠재적 인 유물과 결함을 발견했습니다. 유망한 전임상 데이터가 자주 얻을 수있다하더라도, 실제로, 이들은 거의 중요한 임상 이득으로 변환하지 않습니다. 이 ?...

<table border="1">
	<tbody>
		<tr>
			<td>Regeant</td>
			<td>Company</td>
			<td>Catalog No.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>10X HBSS</td>
			<td>Eurocalne</td>
			<td>ECM4006XL</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peni/Strepto</td>
			<td>Lonza</td>
			<td>DE17602E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Gibco</td>
			<td>15750-037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12614</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS-Na</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100X ITS-X</td>
			<td>Gibco</td>
			<td>51500-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Tebu-bio</td>
			<td>100-15</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Lonza</td>
			<td>BE17605E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non essential aminoacids 100X</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaPyr</td>
			<td>Gibco</td>
			<td>11360-039</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>Cloning cylinders 6x8 mm</td>
			<td>BellCo</td>
			<td>2090-00608</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 8x8 mm</td>
			<td>BellCo</td>
			<td>2090-0080</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cloning cylinders 10x10 mm</td>
			<td>BellCo</td>
			<td>2090-01010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Metal grids</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centre well organ culture plate</td>
			<td>BD Falcon</td>
			<td>353037</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical glue (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen Jars</td>
			<td>Home made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen tanks</td>
			<td>Air Liquid Sanit&agrave;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a novel method for the culture and polarized stimulation of human intestinal mucosa explants

몇 가지 모델은 현재 현실적으로 상호 작용의 다양한 일어나는 복잡한 인간의 대장의 미세 환경을 시뮬레이션하기 위해 존재한다. 동시에 실수로 음식을 섭취하는 병원성 미생물에 대하여 효과적인 면역 반응을 설치하는 동안 그들은 음식 항원에 대한 전체 면역 반응을 유도 내성 형성으로 적절한 항상성이 직접 이러한 상호 작용에 따라 달라집니다. 장내 항상성은 점액의 부착 / 분해를 조절하는 미생물 (식품 관련 유익한 박테리아 변종 포함)와 호스트 간의 여러 복잡한 상호 작용을 통해도 보존되어, 상피 장벽 항균 펩타이드의 생산, 그리고 &quot;교육&quot; 인구의 고유, 장내 상주 림프 세포의 tolerogenic 또는 면역 표현형 제어 &#39;를 상피 세포. 이러한 상호 작용은 생체 분석에로 재현하는 지금까지 매우 어려웠을배양 세포주 또는 말초 혈액 단핵 세포를 사용하여. 또한, 마우스 모델은 인간의 창자에 대하여 장 점막 (점액 층 조직, 공생 박테리아 사회)의 구성 요소에 실질적으로 차이가 있습니다. 따라서, 이러한 과민성 대장 증후군, 염증성 장 질환이나 대장 암과 같은 중요한 스트레스 관련 또는 병적 조건을 병원에 데려 수 치료의 다양한 연구가 수행하기 어려운되었습니다. 이러한 문제를 해결하기 위해, 우리는 우리가 편광 방식으로, 호스트 미생물 및 면역 반응에 의해 현장 조화에서 자신을 유지 인간의 창자 점막의 절편을 자극 할 수있는 새로운 시스템을 개발했다. 편광 혀끝의 자극의 결과에 대한 매우 중요 면역 반응을 이끌어. 그것은 반복적으로 그들이 장 EPI의 정점 얼굴을 무시할 때 동일한 자극이 완전히 다른 반응을 일으킬 수 것으로 나타났습니다thelium, basolaterally 상피 세포를 자극하거나 고유 층의 구성 요소와 직접 접촉으로 오는 tolerogenic에서 면역 원성에 대한 표현형을 전환하고 지역에서 불필요하고 과도한 염증을 일으키는 원인. 우리는 접착에 의해 점막의 정점 얼굴에 자극의 영역이 같은 프로토콜에 설명한다 구분 동굴 실린더 편광 자극을 달성했다. 우리는 다른 세 가지 유산균 균주의 차등 효과 사이 검토하기 위해이 모델을 사용했다. 우리는이 모델 시스템이 건강과 질병 상태에서 프로바이오틱스의 면역 특성을 평가하는 매우 강력한 것을 보여줍니다.

우리는 조직의 웰빙을 유지 적어도 24 시간 동안 문화 건강하고 IBD 인간의 창자 점막을 유지에 성공했다. 문화 시간의 대부분 (전체의 85 % 이상) O 2에서 일어났다면 이전의 관찰 1 항에 따라,이 조직이 기존의 인큐베이터에서 24 시간 (그림 2) 생존하지 않았습니다으로 만 가능했다. 우리는 또한 절편의 다양한 반응을 자극 (혀끝 대 기저)과 자극 2,3의 면역 원성?...

Watch this Scientific Journal Video about A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants at JoVE.com

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

우리는 적어도 24 시간 동안 자극의 다양한 종류에 대한 응답의 문화와 모니터링 인간의 장 점막의 유지 관리를위한 새로운 방법을 소개합니다. 우리의 방법으로, 조직의 극성이 혀끝의 경로를 통해 생리적 자극 수 있도록 유지됩니다.

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 ...

a-novel-method-for-culture-polarized-stimulation-human-intestinal

Research

JoVE Journal

Biology

13.0K 조회수.  European Institute of Oncology. 우리는 적어도 24 시간 동안 자극의 다양한 종류에 대한 응답의 문화와 모니터링 인간의 장 점막의 유지 관리를위한 새로운 방법을 소개합니다. 우리의 방법으로, 조직의 극성이 혀끝의 경로를 통해 생리적 자극 수 있도록 유지됩니다.

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

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 ...

연구

생물학

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

터미널 차별 유학을위한 준비 및 랫 렌즈 상피 Explants의 문화

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

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

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

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

회전 형 바틀 문화 시스템을 사용 로던트 전체 엠브료 문화의 방법

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

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 ...

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

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

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

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

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

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

서브 클로닝 플러스 삽입 (SPI) - 유전자 타겟팅 벡터의 신속한 건설 소설 Recombineering 방법

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

렌티 바이러스 형질 도입 및 장내 Organoids의 다운 스트림 분석을위한 프로토콜

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

색채 금 나노 입자 분석에 적용 구조 전환 앱 타머를 선택하는 방법

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

쥐 대장 점막의 이외에 Microdissection을위한 냉동 절편 방법

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Methods Article

인간의 장내 점막의 Explants의 문화와 편광 자극을위한 새로운 방법