这项研究是由美国心脏协会授予0950118G.to MC - W支持部分。

1。准备抗PECAM - 1抗体偶联的磁珠（磁珠） <ol><li>准备通过使用一个旋涡，直到BSA溶解，混合在50毫升PBS 50毫克BSA的0.1％牛血清白蛋白的PBS。 0.22微米注射器过滤器过滤消毒。保存在4 ° C。 </li><li>在TC罩，转移到1.5 mL Eppendorf管200μL重悬羊抗鼠IgG磁珠，洗珠：山管货币政策委员会，并让坐珠，让泥沙约1分钟。吸上清，去除MPC管和1毫升无菌0.1％BSA / PBS重悬珠。移液器向上和向下重悬珠。 </li><li>重复洗三次，共有四个清洗。 </li><li>删除MPC管和500微升0.1％BSA / PBS重悬珠。 </li><li>管中加入10μL大鼠抗小鼠PECAM - 1（CD - 31）抗体。 </li><li>翻滚在寒冷的房间或在室温下2小时，在4 ° C过夜。 </li><li>用无菌0.1％BSA / PBS清洗珠如在步骤1.2四次中所述。 </li><li>删除MPC管和200微升0.1％BSA / PBS重悬珠。商店抗PECAM - 1抗体偶联的磁珠在4 °长达1个月的彗星。 </li></ol> 2。隔离鼠标从新生小鼠肺血管内皮细胞继续组织净化协议之前，IACUC委员会的批准程序是必需的。 <ol><li>准备好以下几点： <ul><li> 15毫升1毫克/毫升C / D DMEM液。过滤用0.22微米注射器过滤器和加热到37 ° C。 </li><li> 50 ml锥形管15毫升的DMEM，储存在冰</li><li>分离介质（IM），含20％胎牛血清和1x青霉素/链霉素的DMEM </li><li> 2％的明胶。混合2克牛皮肤明胶日水100毫升，高压灭菌15分钟，并储存于4 ° C。暖至37 ° C液化涂装前TC烧瓶。 </li></ul></li><li>麻醉三个6-8天使用注射氯胺酮（100毫克/公斤）和甲苯噻嗪（10毫克/公斤）的IP岁的幼崽。麻醉效果，并解剖动物逐一检查，如下：板引脚的幼崽，用70％乙醇浸泡的皮肤和去除胸部皮肤，用无菌剥离剪刀。海关暴露肺部肋骨。 </li><li>无菌消费个体肺癌的肺叶，小心不要解剖任何可见支气管和肺部周围的结缔组织。结合在冰冷的DMEM所有肺部。安乐死的幼崽。 </li><li>工作训练班罩，肺部肺叶和删除的DMEM，在100毫米训练班菜的地方，吸出多余的DMEM。 </li><li>使用无菌剪刀，肉组织精细切割〜100倍。转移至15毫升暖C / D酶消化解决方案。在37 ° C孵育45分钟，一个肩。 </li><li>在TC发动机罩，吸14克导管连接和磨碎成单细胞悬液的团块，至少有12次到20毫升注射器消化的组织悬液。 </li><li>穿过70微米的细胞过滤器组织悬液，用15毫升的IM过滤器停止消化。 </li><li>颗粒细胞悬液，离心5分钟400X克。 </li><li>吸上清，重新悬浮颗粒在3毫升0.1％的BSA / PBS。 </li><li>将细胞悬液5 ml圆底聚苯乙烯管，并添加22.5μL抗PECAM - 1抗体偶联的磁珠。翻滚在室温为12分钟。 </li><li>外套与T75烧瓶2 mL 2％的明胶和吸出多余的明胶。允许明胶训练班罩内的干燥约30分钟前电镀细胞。 </li><li>珠孵化完成后，（步长2.10），分裂细胞悬液分为3个Eppendorf管，并安装在MPC的。 </li><li>当珠使用MPC（〜1分钟）沉淀，收集上清，去除MPC管，洗珠〜1毫升0.1％的BSA / PBS，然后重新安装的MPC。 </li><li>重复洗四次（共5洗） </li><li>重悬珠EnGS - MV寿命的因素套件和1x青霉素/链霉素（VL），每管1毫升VascuLife，搅拌好。 </li><li> T75烧瓶中，预涂层钢板，并在每个烧瓶中带来的总体积至10毫升与VL。 </li><li>更改媒体翌日，允许增长1整天，然后更改隔日媒体的一半。当细胞&gt; 70％-80％汇合或多个（通常在3-4天的文化），他们可以与抗ICAM - 2磁珠进行排序。 </li></ol> 3。准备抗ICAM - 2的抗体偶联的磁珠<ol><li>抗ICAM - 2的抗体偶联的磁珠用于进一步净化抗PECAM - 1抗体偶联的磁珠和培养，直到融合已选定的单元格。抗PECAM - 1抗体偶联的磁珠（1段）所述，除，抗小鼠ICAM - 2（CD - 102）抗体用于抗PECAM - 1抗体，而不是执行此过程。 </li><li>抗ICAM - 2的抗体偶联的磁珠在4 ° C，最多可存储1个月。 </li></ol> 4。排序小鼠肺血管内皮细胞与抗ICAM - 2磁珠<ol><li>大衣T75组织2％明胶的培养瓶中。 </li><li>吸媒体与细胞融合T75烧瓶，用8毫升PBS。 </li><li>吸PBS中，加2毫升0.05％胰蛋白酶/ EDTA，让细胞分离（约5分钟）。轻轻点击烧瓶，以帮助分离。 </li><li>当细胞分离，加入2 ml的IM抑制胰蛋白酶和刮细胞分离任何剩余的贴壁细胞。将细胞悬液15毫升管和自旋为5分钟400X G. </li><li>在2毫升0.1％的BSA / PBS吸媒体和重悬细胞沉淀。 </li><li>转移到5毫升的聚苯乙烯圆底管，添加10μL抗ICAM - 2磁珠。翻滚在室温为12分钟。 </li><li>拆分成两个1.5 ml Eppendorf管中，并装入MPC细胞悬液。 </li><li>珠坚持一分钟后，吸上清液。删除MPC管，每管1毫升0.1％的BSA / PBS重悬。重新装入对货币政策委员会。 </li><li>重复洗4次（共5洗）。 </li><li>重悬每管1毫升VL，并进行细胞计数。 </li><li>在250 - 350K电池板每T25烧瓶和/或750K - 1万个细胞T75烧瓶细胞。 </li><li>带量5毫升VL的T25烧瓶中，在T75烧瓶和10毫升。 </li><li>更改媒体隔日。文化达到约80％汇合，通常在2-3天，的细胞可用于实验。 </li></ol> 5。代表性的成果通常情况下，从细胞的前期准备工作后6-7天，我们能够获得约1.2 - 1.5 × 10 6从三个6-8日龄仔兔肺血管内皮细胞。细胞显示典型的“鹅卵石”形态和光镜下显示的VE -钙粘蛋白（CD144）在细胞与细胞交界处的染色，这是对内皮细胞的特征（图1）。 MLECs表达VE - cadherin和VEGFR2的大部分证明用流式细胞仪分析（图2）。通常，我们使用后在两个星期内的实验，他们最初的“电子商务示范法”第隔离。 <img src="/files/ftp_upload/2316/2316fig1.jpg" alt="图1" /> 图1。微观分析融合“电子商务示范法”第单层。 （一）光学显微镜图像显示了典型的内皮细胞培养的细胞形态的鹅卵石。在许多细胞的核周区位于统一一轮结构“电子商务示范法”第隔离使用的磁珠。 （二）使用共聚焦显微镜所显示的细胞 - 细胞连接处的内皮具体的VE - cadherin的本地化。酒吧是20μm的代表。 <img src="/files/ftp_upload/2316/2316fig2.jpg" alt="图2" /> 图2流式细胞仪的标记抗体对内皮细胞的特异性标志特定的培养MLECs 分析 ：VE -钙粘蛋白（CD144）或VEGFR2的，如被起诉，藻红蛋白标记的二抗，内皮身份确认孤立MLECs 。红线表示亚型具体的控制，绿线表示反VE - cadherin的或抗VEGFR2的 - 特定抗体。

<ol>
	<li>Maruyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+endothelial+cell+in+tissue+culture.">The human endothelial cell in tissue culture.</a> Z Zellforsch Mikrosk. Anat. 60, 69-79 (1963).</li><li>Lim, Y. C., Luscinskas, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+murine+heart+and+lung+endothelial+cells+for+in+vitro+model+systems.">Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.</a> Methods Mol Biol. 341, 141-154 (2006).</li><li>Aird, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity+of+the+endothelium:+I.+Structure,+function,+and+mechanisms.">Phenotypic heterogeneity of the endothelium: I. Structure, function, and mechanisms.</a> Circ Res. 100, 158-173 (2007).</li><li>Fehrenbach, M. L., Cao, G., Williams, J. T., Finklestein, J. M., Delisser, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+murine+lung+endothelial+cells.">Isolation of murine lung endothelial cells.</a> Am J Physiol Lung Cell Mol Physiol. 296, L1096-L1103 (2009).</li><li>Miebach, S., Grau, S., Hummel, V., Rieckmann, P., Tonn, J. C., Goldbrunner, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+microvascular+endothelial+cells+from+gliomas+of+different+WHO+grades.">Isolation and culture of microvascular endothelial cells from gliomas of different WHO grades.</a> J. Neurooncol. 76, 39-48 (2006).</li><li>Demeule, M., Labelle, M., R&#233;gina, A., Berthelet, F., B&#233;liveau, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+brain,+lung,+and+kidney:+expression+of+the+multidrug+resistance+P-glycoprotein+isoforms.">Isolation of endothelial cells from brain, lung, and kidney: expression of the multidrug resistance P-glycoprotein isoforms.</a> Biochem. Biophys. Res. 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动物实验是由威斯康星IACUC委员会根据协议AUA＃00001206医学院规定的准则和法规的规定执行。

微血管内皮细胞已被证明是一个有用的模型在血管生物学的许多领域，被认为是有关生理研究比 3脐静脉内皮细胞广泛的研究（如血管） 。此前，有报道，微血管内皮细胞可以从肾脏，心脏，皮肤，视网膜，脑，神经胶质瘤，脂肪组织和肺2，4-7，10，11获得。然而，微血管内皮细胞的隔离的一致和可靠的方法仍然是必需的。这里介绍的程序是以前发布的协议2的修改;从最公布的程序，它使用了幼崽，而不是成年动物的不同。这是隔离成功的关键，年轻动物的细胞有较高的增殖潜能和来自其组织的文化，往往会产生较高的细胞数量。一个星期的动物似乎是最佳的，但年轻的老鼠（4日龄）也可以使用。此外，可以使用较高或较低的幼崽，如果需要的话，因为我们已经成功隔离MLECs从一个小狗。然而，同时缩​​放向上或向下的隔离过程中，细胞电镀密度必须保持不变，因为这也是细胞增殖的关键。首先使用共轭磁珠抗PECAM - 1和抗ICAM - 2的抗体比MLECs 2步纯化过程，是更有效的获得纯EC比前面描述的单步纯化过程文化。此协议消除了需要使用很费力的手工技术，梯度离心法和分类丢弃污染的细胞，如成纤维细胞，血细胞和平滑肌细胞的效率较低，流式细胞仪。 MLEC人口所产生的可用于在体外培养的血管生成响应，血管通透性和白细胞轮回分析，伤口愈合以及信号转导通路的生化分析。如果需要，MLECs可镀上不同的表面，如免疫或跨内皮电阻（TER）测量电极阵列的纤维连接蛋白或明胶包被的盖玻片。可以直接得到的结果相比， 在体内 ，这一点特别重要，在越来越多的基因敲除和转基因小鼠线背景下观察到的表型。成功地实施这种方法，在体外体内观察到的缺陷的细胞机制的分析，已提出12。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15032-13</td>
<td>&nbsp;</td>
<tr>
<td>forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11223-20</td>
<td>&nbsp;</td>
<tr>
<td>14G cannula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482516N</td>
<td>&nbsp;</td>
<tr>
<td>20 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309661</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich </td>
<td>A7030-100G</td>
<td>&nbsp;</td>
<tr>
<td>anti-rat IgG Dynabeads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>110.35</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Particle Concentrator (MPC)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>123-21D</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse PECAM-1 antibody (CD-31)</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553369</td>
<td>&nbsp;</td>
<tr>
<td>anti-mouse ICAM-2 antibody (CD-102) </td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>553326</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase/Dispase (C/D)</td>
<td>&nbsp;</td>
<td>Roche Applied Science</td>
<td>11097113001</td>
<td>100mg/ml stock solution can be prepared and stored at -20&deg;C</td>
</tr>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro Mediatech</td>
<td>10-013-CV</td>
<td>&nbsp;</td>
<tr>
<td>Bovine Skin Gelatin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>#G9391-100G</td>
<td>&nbsp;</td>
<tr>
<td>Vasculife EnGS-Mv complete kit (VL)</td>
<td>&nbsp;</td>
<td>Lifeline Cell Technology</td>
<td>LL0004</td>
<td>&nbsp;</td>
<tr>
<td>0.05% Trypsin/EDTA </td>
<td>&nbsp;</td>
<td>Mediatech</td>
<td>25-052-CI</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer 70 &mu;m nylon</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>tissue culture flask 75 cm2</td>
<td>&nbsp;</td>
<td>TPP</td>
<td>90076</td>
<td>&nbsp;</td>		</tbody>
		</table>
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isolation and culture of pulmonary endothelial cells from neonatal mice

内皮细胞在血管生物学的许多领域提供了有益的研究模型。自第一次隔离1，人脐静脉内皮细胞（内皮细胞）要方便，容易获得和文化，因此是研究最广泛的内皮细胞。然而，对于重点研究的血管生成，透气性好或许多其他的进程上，微血管内皮细胞（ECS）是一个更有关生理模型 ，研究2。此外，内皮细胞基因敲除小鼠分离蛋白质功能的体外分析提供了一个有用的工具。几种方法来隔离和文化微血管内皮细胞对不同产地已上报日期3-7，但一致的纯内皮细胞的分离和文化仍然是一个在许多实验室的主要技术问题。在这里，我们提供一个可靠和相对简单的方法分离和培养小鼠肺血管内皮细胞（MLECs）一步一步的协议。在这种方法中，肺组织6 - 8岁的幼崽首次切成片，与胶原酶/ dispase（C / D）解决方案，消化和分散成单细胞悬液机械一天。 MLECS纯化细胞悬液，使用抗PECAM - 1抗体结合使用磁粉集中器（MPC）的磁珠阳性选择。这种纯化的细胞培养明胶涂层的组织培养（TC）菜，直到他们成为融合。在这一点上，细胞进一步纯化，再加上以抗ICAM - 2抗体的磁珠。本议定书获得MLECs展示一个鹅卵石的表型，相衬光学显微镜观察，并已被证实使用反VE - cadherin的8和抗VEGFR2的抗体和VE - cadherin的免疫荧光染色流式细胞仪分析其内皮细胞表型。在我们手中，这两个步骤的分离过程中稳定，可靠的收益率的MLECs纯粹的人口，这可以进一步培养。这种方法使研究人员利用基因敲除和转基因小鼠体内研究直接相关，在对孤立MLECs 进行的体外实验结果越来越多的优势， 从而有助于揭示体内观察到的血管表型的分子机制。

Watch this Scientific Journal Video about Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice at JoVE.com

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

在这里，我们描述了一个小鼠肺血管内皮细胞的分离和培养协议。这种方法包括机械和酶的肺组织分解以及2步纯化过程中使用抗PECAM - 1和抗ICAM - 2的抗体结合磁珠，这将产生一个纯粹的原籍大多微血管的内皮细胞的人口。

从新生小鼠的肺血管内皮细胞的分离和培养

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

isolation-culture-pulmonary-endothelial-cells-from-neonatal

Research

JoVE Journal

Biology

46.9K 次观看.  BloodCenter of Wisconsin. 在这里，我们描述了一个小鼠肺血管内皮细胞的分离和培养协议。这种方法包括机械和酶的肺组织分解以及2步纯化过程中使用抗PECAM - 1和抗ICAM - 2的抗体结合磁珠，这将产生一个纯粹的原籍大多微血管的内皮细胞的人口。

视频: Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

瓣膜内皮细胞的分离

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

科研

生物学

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

分离新生小鼠心肌文化和

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

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.

胚胎心室血管内皮细胞的分离

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

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

肺动脉平滑肌细胞从新生小鼠隔离

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

隔离和鼠标主动脉内皮细胞原代培养

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

人外周血代文化血液的产物内皮细胞

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

鼠标冠状动脉内皮细胞的分离

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

来自新生儿和成年鼠皮肤的原代小鼠角化细胞的分离和培养

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...