현재 프로토콜의 개발을위한 자금 조달은 특히 국립 대만 대학, 대만 (부여 ID-104R350144)뿐만 아니라 대만의 과학 기술부 (부여 ID &quot;상위 대학 계획에 대한 목표&quot;에 의해 지원됩니다 MOST (104) -2313-B-002-039-MY3). 우리는 특히 현재의 연구를위한 동물 실 및 실험실 공간을 제공하기 위해 국립 대만 대학의 생명 공학 센터 감사합니다.

참고 :..이 절차는 등 바우어 등 2013 나카자와 등, 2011 년 2,9에 의해 개발 된 치킨 모델 문화 프로토콜의 수정입니다 1. 건강한 배아 5 일 태아 일본어 메추라기에서 준비 <ol><li> 같은 케이지에서 함께 한 남자와 세 여성의 성적으로 성숙한 일본어 메추라기를 놓습니다. 전원 공급 및 임의 량의 물. 빛의 14 시간과 어둠의 10 시간에 동물 방에 빛을 조정합니다. </li><li> 오후에 매일 비닐 봉지에 수정 된 알을 수집하고 태아의 성장 속도를 지연하기 위해 16 ° C 냉장고에 보관하십시오. </li><li> 2 주 기간 내에 계란의 충분한 수를 수집 한 후, 5 일 동안 통풍이 잘되는 37 ° C 배양기에서 수정 된 알을 품어. </li></ol> 2. 기초 배지 및 세척 솔루션을 준비 <ol><li> 성장 배지로 (pH를 7.2로 조정) DMEM / F-12 배지를 사용하고, 그것을 보충10 % 새로 태어난 송아지 혈청 (NBCS), 1 % 펜 연쇄상 구균 Ampho와. 솔루션 (PSA; 페니실린, 105 단위 / L, 스트렙토 마이신, 100 ㎎ / ℓ, 암포 테리 신 B, 0.25 ㎎ / ℓ). </li><li> 저장을위한 10 배 농도의 인산 완충 식염수 (PBS) 용액을 제조; 씻어 (pH를 7.2로 조정;의 KCl, 2.667 밀리미터, KH 2 PO 4, 1.471 밀리미터; 나 2 HPO 4 -7H- 피 2 O, 8.06 mM의 NaCl을, 137.93 밀리미터 포함)과 난황 막 동안 축축하게 1X PBS 솔루션을 사용 계란 노른자와 알부민에서 내배엽을 분리. </li><li> 내배엽 펠렛을 다시 중단 (pH를 7.2로 조정) DMEM 배지를 사용합니다. </li></ol> 일본어 메추라기 YSMs에서 차 EECS 3. 컬렉션 <ol><li> 오일 (단계 1.3)에 대한 수집 한 알을 품어. 정상 배아 발달을 확인 달걀 ​​캔 들러에 의해 계란을 검사합니다. 참고 : 배아 일 5 일 YSM의 중배엽의 경계는 매우 명확하고 3 배아 층은 덜 함께 밀접하게 연결되어있다, 그래서 OB하기 쉬운주석 박의 내배엽. 만 YSM 컬렉션 정상적인 모세 혈관 순환 개발을 보여주는 계란을 사용합니다. </li><li> 주의 깊게 신선한 물과 75 % 에탄올을 사용하여 껍질을 청소합니다. 부드럽게, 가위로 공기 셀 위치에서 달걀 껍질을 열고 밖으로 껍질, 37 ° C 1X PBS로 가득 10cm 배양 접시에 집게와 장소에 의해 전체 YSM를 수집합니다. </li><li> 가위는 배아, 난황과 난백을 분리하고 부드럽게 1X PBS로 세척 YSM 세 번 및 10cm 배양 접시에 PBS 용액 YSM 배치하여. </li><li> YSM에서 외배엽을 제거하기 위해 집게와 가위를 사용합니다. 집게를 사용하여 2 번 단단히 내배엽 세포 층을 보유하고, 다른 하나는 모세관 중배엽을 잡아. </li><li> 해부 현미경에서 떨어져 당겨 배아를 향한 방향으로 중배엽의 가장자리에서 내배엽를 구분합니다. 37 ° C 와트 성장 배지 15 ㎖ 중의 적기 장소를 이용하여, 50 ml 원심 분리 튜브 내에서 담황색 내배엽 모아서어 목욕 모든 조직 모음이 완료 될 때까지. </li></ol> 콜라게나 소화에 의해 내배엽 조각 4. 소화 <ol><li> 갓 DMEM 10 ml의 유형 IV 콜라​​게나 6.5 단위를 녹인다. </li><li> RT에서 3 분 동안 130 XG에 50 ㎖ 튜브 (3.4)을 원심 분리기. 매체를 기음과 피펫을 사용하여 6cm 배양 접시에 모든 내배엽을 배치합니다. 이 배양 접시에 1 ml의 콜라게나 솔루션을 추가합니다. </li><li> 각 조각의 크기까지 곡선 가위 2 메추라기 배아에서 수집 내배엽 슬라이스 대략 2-3mm이다. , 적기를 사용하여 신선한 콜라게나 솔루션의 9​​ ml의와 함께 50 ㎖ 원심 분리 튜브에 모든 조각을 수집합니다. </li><li> 175 rpm에서 37 ° C 진탕 수조에서 30 분 동안 콜라게나 조직 다이제스트. 콜라겐이 부분 소화 이식편의 세포 증식을 향상시키는 데 사용된다. </li><li> RT에서 3 분 동안 130 XG에 원심 분리기 부드럽게 피펫을 사용하여 상층 액의 일부를 제거. 20 ㎖ DMEM에 현탁하여 펠렛을 씻고 3 분 130 XG에 원심 분리 후 상층 부분을 제거합니다. </li><li> 다시 중단 성장 매체 (10 % NBCS 1 %의 PSA와 DMEM / F12)의 12 ml의와 펠렛을, 24 웰 플레이트에 부드럽게와 동일 (각 웰에 500 ㎕의 세포 솔루션을) 시드. </li><li> 세포 조직에서 증식 할 수 있도록 2 일 동안 공기 중에서 5 %에서 37 ° C에서 CO 2 조직 이식편 부화. </li><li> 추가 2 일 동안 세포를 인큐베이션하고 세포 생존 분석 프로토콜에 의해 세포 생존율에 대한 특성화. 15 주 : 살아있는 세포는 세포질 내에서 환원 환경을 유지한다. 레사 주린, 활성 성분은 푸른 색과 거의 비 형광 비 독성 세포 투과성 화합물이다. 세포 배양에 첨가 될 때, 레사 주린은 미토콘드리아 효소에 의해 세포질로 레조 루핀 (resorufin)로 감소된다. 레조 루핀 (resorufin)는 붉은 색과 높은 형광입니다. 실행 가능한 세포가 지속적으로 레사 주린은 레조 루핀 (resorufin)로 변환, incre주변 세포 배양 배지의 전체 형광 색 asing. </li><li> 한 바와 같이 전사 및 PCR 마커 유전자의 mRNA 발현의 정량적 실시간 역 내배엽 상피 세포의 총 RNA 분리를 수행한다. (16, 17)을 <ol><li> SOAT1 mRNA를 검출하는 역전사 효소를 사용하여 PCR (센스 : 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;안티센스 : 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR 생성물을 B​​P = 168) EEC 세포 특이 마커로. 하우스 키핑 유전자 및 내부 통제로 β-굴지의 mRNA (PCR 제품 = 151 bp의 : &#39;; 5&#39; GTGATGGACTCTGGTGATGG-3 안티센스&#39;5&#39;- TGGTGAAGCTGTAGCCTCTC-3 감각)를 사용합니다. </li><li> RT-qPCR에 제품의 크기를 검출하는 겔 전기 영동을 사용한다. 1 % 아가 로스 젤을 준비, 2 μL 로딩 염료 10 μl의 qPCR에 제품을 혼합한다. 로드 10 μL 웰을 1 % 아가 로스 겔에서 각 혼합물에, 30 분 동안 100V로 겔을 실행. </li></ol></li></ol>

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There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

전 배양 시스템은 제한된 성공을 갖기 때문에, 더 배양 시스템이 필요하다. 일본 메추라기 YSM의 내배엽은 전 생체 내 외식의 더 나은 성장 성능을 달성하기 위해 세포 - 세포 접합을 완화하기 위해 콜라겐 등의 단백질 분해 효소와 치료가 필요합니다. 우리의 데이터는 부분 소화 처리에서 세포 수는 이일 (그림 1)에 대한 시딩 후 소화되지 않은 조직 문화에서보다 더 컸다 것으로 나타?...

조류 배아의 주요 영양 자원이 33 % 지질, 17 %의 단백질로 구성 노른자, 1 % 재. 1 배아 개발 중입니다, 난황 막 (YSM)는 배아 복강 내에서 성장하고 점차적으로 노른자를 커버 표면. 배아 2 일에 시작하여, 지질 대사 및 혈관 신생과 관련된 유전자의 발현이 점차 YSM 증가되고 YSM 천천히 융모와 같은 돌기를 발생한다. -8,9- 이러한 돌기는 배아 발달을 지원하기 노른자 영양소의 흡수를 증가시킨다. YSM 세 가지 배엽, 내배엽, 중배엽과 외배엽을 포함하는 배외 (extraembryonic) 조직이다. (14) 난황 외배엽 부드럽게 난황을 포함하는 난황 막과 알부민 및 링크에 직면 해있다. 내배엽 상피 세포는 shap로 나눌 수있다 달걀 노른자를 향해 직접 직면하고 영양 활용 포털의 역할을한다. 6 YSM이 확장하면서, 내배엽 상피 세포 (EECS)를하고 있습니다두 그룹, 지역 난황 및 지역 vasculosa에 전자 및 기능을 제공합니다. (7) 영역 난황는 내배엽 세포로 구성되며 배아로부터 떨어진되고 영역 vasculosa는 중배엽 세포로 구성되고, 혈관 결합 조직으로 분화 EECS을 덮는다. 배아 5 일까지, 노른자는 완전히 YSM의 외배엽과 내배엽이 적용되고, 혈관 영역은 빠른 속도로 성장하고있다. YSM은 배아 순환 시스템으로 (노른자 유래 매우 저밀도 지질 단백질 등)과 단백질. 9, 2 따라서, 우리는 지질의 메커니즘을 연구하기 위해, 기본 일본어 메추라기 배아 내배엽 상피 세포 배양 시스템을 구축 recomposes 해제 지질 흡수 조류 배아 개발하는 동안 YSM에 활용. 예컨대 중성 지방, 레시틴, 인지질 및 콜레스테롤 에스테르 (CE)와 같은 지질 조류 배아의 주 에너지 원이다. 개발의 초기 단계에서, 난황 지질 C는1.3 %의 CE의 omposed하고 조류 배아 발달 (3)의 중간 10-15 %로 상승, 11. 콜레스테롤 에스테르 조류 배아 YSM에 스테롤 O-아실 트랜스퍼 라제 1 (SOAT1)에 의해 콜레스테롤로부터 합성된다. (4) 콜레스테롤의 저장 형태는 CE, CE는 지질 단백질에 실시하고, 지질 단백질은 조직에 혈액 순환에 의해 수송된다. 13 주 해치 전에 조류 배아의 급속한 성장이있다. 약 노른자의 나머지 지질 함량의 68 %는이 단계에서 흡수된다. 10 지질 EEC 연구 모델로 정화 할 수있다 이용된다 노른자하는 메커니즘. 닭 EEC 배양 프로토콜은 본 연구의 목표를 ​​달성하기 위해 설립되었다. 2, 9, 그러나, 향상된 EEC 세포 배양 방법에 의해 영양분 활용도를 매개 특정 효소와 단백질의 기능을 연구하는데 필요한 조직 이식편의 낮은 성공률로 개발하는 동안 조류 배아.

<table border="0" cellpadding="0" cellspacing="0" width="845">
	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12800-017 10 X 1 L</td>
			<td style="width:167px;">For wash the EECs pellets</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">D-MEM/F-12</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">12400-024 10 X 1 L</td>
			<td>As the basal medium in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">NBCS</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">16010-159</td>
			<td>As the supplyment serum in culturing EECs</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Pen-Strep Ampho. Solution</td>
			<td style="width:199px;">BI (Biological Industries)</td>
			<td style="width:148px;">03-033-1B 100ml</td>
			<td>For attenuating the possible infection&#160;</td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:331px;">Collagenase Type IV</td>
			<td style="width:199px;">Gibco by Life technologies</td>
			<td style="width:148px;">17104-019 1g</td>
			<td>&#160;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">24 well plate</td>
			<td style="width:199px;">FALCON&#174;</td>
			<td style="width:148px;">REF-353047</td>
			<td>For EECs to attach and extension</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:331px;">50 ML PP centrifuge tubes</td>
			<td style="width:199px;">Corning&#174; CentriStarTM</td>
			<td style="width:148px;">430829</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">50ML Conical bottomed Tube with Cap</td>
			<td style="width:199px;">PRO TECH</td>
			<td>CT-50-PL-TW</td>
			<td>For transportion of membranes and enzyme digestion</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Reciprocal shaking bath</td>
			<td>DEAGLE</td>
			<td style="width:148px;">SB302</td>
			<td>For better enzymatic digestion on membranes</td>
		</tr>
	</tbody>
</table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

우리는 개발 과정에서 조류 배아에 의해 영양소 이용률을 매개하는 특정 효소와 단백질의 기능을 연구하는 내배엽 상피 세포 배양 모델 (EEC)를 설립했다. 수정 된 일본어 메추라기 계란 5 일 동안 37 ° C에서 배양 한 다음 골목 막 (YSM)는 EEC 문화 시스템을 구축하기 위해 수집 노른자 하였다. 우리 YSM에서 배아 내배엽 층을 단리하고, (2) 내에 막 분리 - MM 3 개의 부분적으로 24 웰 배양 플레이트에 파종 전에게나로 분해. EECS는 조직에서 증식 및 세포 배양 연구를위한 준비가되어 있습니다. 우리는 EECS 예를 들어 생체 내에서 YSM의 일반적인 특성, 지질 방울의 축적, 스테롤 O-아실 트란스 퍼과 지단백 리파제의 발현을 한 것으로 나타났습니다. 부분 소화 치료는 크게 EEC 문화의 성공적인 속도를 증가했다. EECS을 활용, 우리는 SOAT1의 발현이 캠프 D에 의해 규제 것을 증명ependent 단백질은 관련 경로를 키나아제. 이 차 일본어 메추라기 EEC 문화 시스템은 배아 지질 수송을 연구하고 조류 배아 개발하는 동안 YSM 영양소 이용률을 매개에 관여하는 유전자의 역할을 명확히하기 위해 유용한 도구입니다.

일관성 있고 유용한 셀 모델을 설정하는 목표를 달성하기 위해, 우리는 확장 증식 속도 및 조류 EECS의 성능을 안정화 할 필요가있다. 우리는 부분적 콜라겐 또는 콜라겐 플러스 디스 파제 0.6 U와 같은 단백질 분해 효소로 분해 내배엽 아니 효소 소화와 내배엽의 직접 배양을 비교 하​​였다. 디스 파제는 비극성 아미노산 잔기의 N 말단 펩티드 결합을 가수 분해 아미노 - ?...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

일본 메추라기 배아의 노 른 골목 막에서 차 내배엽 상피 세포 배양

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Research

JoVE Journal

Developmental Biology

8.8K 조회수.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

비디오: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

차 세포 배양 연구 병아리 망막에 효율적인 유전자 전송 :<em&gt; 전 비켜</em&gt; Electroporation에 접근

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

연구

발생학

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

분리 및 Zebrafish의 배아에서 단일 세포의 특성

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

고도의 재현 작은 분자 기반의 차별화 프로토콜을 사용하여 인간 다 능성 줄기 세포로부터 심근 세포의 대규모 생산

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Zebrafish의 배아에서 내인성 수용체의 친화력 레이블 검출

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Whole-cell Patch-clamp Recordings of Isolated Primary Epithelial Cells from the Epididymis

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types that are distinct not only in molecular and morphological features but also in physiological properties. These differences reflect their diverse functions, which together establish the necessary microenvironment for the post-testicular sperm development in the epididymal lumen. The understanding of the biophysical properties of the epididymal epithelial cells is critical for revealing their functions in sperm and reproductive health, under both physiological and pathophysiological conditions. While their functional properties have yet to be fully elucidated, the epididymal epithelial cells can be studied using the patch-clamp technique, a tool for measuring the cellular events and the membrane properties of single cells. Here, we describe the methods of cell isolation and whole-cell patch-clamp recording to measure the electrical properties of primary dissociated epithelial cells from the rat cauda epididymides.

We present a protocol that combines cell isolation and whole-cell patch-clamp recording to measure the electrical properties of the primary dissociated epithelial cells from the rat cauda epididymides. This protocol allows for investigation of the functional properties of primary epididymal epithelial cells to further elucidate the physiological role of the epididymis.

부고환에서 분리 된 1 차 상피 세포의 전체 세포 패치 - 클램프 기록

The epididymis is an essential organ for sperm maturation and reproductive health. The epididymal epithelium consists of intricately connected cell types ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

마우스 Preimplantation 배아에서 줄기 세포 라인의

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM와 Microrheology Zebrafish 태아 노른자위 셀에

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

대동맥-생식-mesonephros Explant 문화 시스템 개발 조에의 응용

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

분리와 인간 성인 그리고 태아 심장 표본에서 파생 하는 기본 Epicardial 세포의 문화

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...