我们感谢俄亥俄州立大学综合癌症中心的流式细胞仪分析核心设施，特别是卡特里娜·摩尔，用于FACS分析技术援助。此外，我们认识到浦威廉博士和他的研究小组对他们的科学见解。这项工作是由美国国立卫生研究院HL091878（JL）和心脏中心在全国儿童医院的支持。

1，前处理设备和解决方案<ol><li>消毒清扫工具 - 细剪的组织中提取的成人心和2细镊子的瓣膜区的清扫 - 在有盖的仪表盘通过高压灭菌。喷用70％乙醇（EtOH中）之前清扫工具。 </li><li>准备好所有的解决方案在紧接实验，通过无菌0.2微米的过滤器将它们消毒解决方案。保持在冰上的解决方案，直到使用。 <ol><li> 无菌分离缓冲液 （15毫升总/样品）。结合1.2毫升Ⅳ型胶原酶，300微升2.5％胰蛋白酶和150微升鸡血清。使体积达15毫升，Hank氏平衡盐溶液（HBSS）中。 </li><li> 无菌排序缓冲区 （12毫升总）。结合25微升的0.5M乙二胺四乙酸（EDTA）和12微升DNA酶I（不含RNA酶）。带量高达12毫升的HBSS。 </li><li> 血管内皮细胞培养基。混合内皮GRO相机连按制造商的说明（见材料的电子表格）通过从所述试剂盒至500ml EBM2媒体的加入等分的组件介质。 </li><li> 绿色荧光蛋白阴性文化传媒 （非内皮细胞介质）。混合445毫升中199 1X与5 ml青霉素/链霉素（青霉素/链霉素）（1％终浓度）和50毫升胎牛血清（10％终浓度）。 </li></ol></li></ol>从成年小鼠E14.5胚胎2解剖瓣膜区<ul><li> 所有动物的程序获得批准，并按照全国儿童医院研究所IACUC准则进行的。 </li><li>的Tie2-GFP的小鼠从杰克逊实验室（股票号003658）14购买并维持纯合基因型上的FVB / N的背景，因此，确保在Tie2的阳性内皮细胞全部脱落弹簧表达绿色荧光蛋白。 </li><li>对于流式细胞仪分选，预备一个年龄匹配的负面SAMP勒不包含GFP如C57 / BL6设置GFP栅参数用于FACS分选。注:本协议和代表性的成果是基于使用三，四月龄的成年小鼠或一窝在胚胎一天（五）14.5。 </li></ul><ol><li>成年小鼠:紧随牺牲的CO 2缺氧，用解剖剪刀轻轻打开胸腔暴露心脏和肺。一旦接触，握钳心脏的大血管和拉离身体。如果完整取出肺组织，和地方的心在冷的HBSS冲洗。取出心室和拉远心房留下房室管&#39;环&#39;和主动脉区的完整。做一个切口向下房室通道的一侧开辟了"环"和揭露瓣膜结构。轻轻用钳子戏弄客场取下心肌和主动脉近端区域。识别瓣叶白，致密组织在粉红色的心肌。轻轻DETA从房室瓣CH腱索和删除尽可能多的剩余心肌越好。一定要小心不要拉扯或在此过程中刮瓣叶，因为血管内皮细胞可脱落。将修剪瓣膜地区，在含有1毫升的HBSS 1.5ml离心管中，并保持在冰上。注意:仔细清扫关键是要消除非瓣膜内皮细胞可能污染样品。 </li><li>胚胎:牺牲雌性小鼠后14.0天交配塞观察（早晨交配塞= 0.5天）。 <ol><li>紧接着牺牲，解剖子宫（含胚胎），并用冷的HBSS。剖析了从子宫单个胚胎并取出周围的每个胚胎的胚胎卵黄囊。删除从每个胚胎的心，按照步骤2.1。 （注意:轻轻挤压用钳子刚上附属物低于胚胎应有助于暴露心脏，使搬迁更加容易。） </li></ol></li></ol><pCLASS ="jove_title"&gt; 3。细胞分离，并准备流式细胞仪<ol><li>用无菌枪头，从包含瓣膜地区的Eppendorf管中取出的HBSS。 </li><li>更换1毫升缓冲液分离和4微升DNA酶一</li><li>旋转试管，在37℃7分钟。 </li><li>移液器上下3次，然后让样品定居，持续15秒。收集上清液（含有解离的细胞）入15ml锥形管中。 </li><li>停止胶原酶反应，加入125微升的马血清至收集管。保持在冰上收集管。 </li><li>重复步骤分解（3.2 - 3.5）的九倍，收集9级上清液。 </li><li>通过70微米尼龙过滤器的分馏收集到一个新的收集管，以确保单个细胞悬液通过消除任何碎片和团块。 </li><li>旋转的悬浮液，在400克5分钟以沉淀细胞。 </li><li>重悬细胞沉淀在1ml排序缓冲器和保持在冰上，直到流式细胞仪。 </li></ol> 4，流式细胞仪分选GFP阳性血管内皮细胞<ol><li>设置闸门的前向和侧向散射以排除碎片和捕捉单个细胞;通过使用双重歧视门排除双峰。记录的阴性对照样品和以对的Tie2-GFP的样品中比较准确地定义了GFP阳性细胞群定义的门限设置。 </li><li>通过流式细胞仪分析GFP阳性细胞和完善的门限设置。 </li><li>排序的Tie2-GFP的样品，并收集两个GFP阳性和GFP阴性细胞。如果细胞将被用于RNA提取，排序直接插入排序缓冲器。以FACS后培养的细胞，收集的GFP阳性细胞在含有1毫升胎牛血清1毫升VEC介质的无菌管中，并收集GFP阴性细胞在1ml的非内皮媒体的用1毫升胎牛血清。使用这个50％的媒体/ 50％血清相结合，提高细胞活力。置于冰上，直到后流式细胞仪分析。 </li></ol> 5，邮政流式细胞仪分析;软件Š <ol><li> RNA提取: <ol><li>紧接FACS，离心机GFP阳性和阴性细胞在1,500×g离心8分钟。 </li><li>小心吸取上清液（排序缓冲区）并丢弃。 </li><li>重悬细胞沉淀（沉淀是不为这里推荐的样本大小可见）在200μl的Trizol。 </li><li>冷冻于-80℃或开始标准的苯酚-氯仿抽提分离总mRNA为先前由本实验室15说明。 </li><li>使用50-200纳克的RNA，用于根据制造商的说明使用Mastermix cDNA合成。 </li><li>受试者的cDNA定量PCR扩增使用IDT黄金时间的qPCR探针针对小鼠内皮细胞标记物（CD31，血管性血友病因子（vWF）的 ），阀间质细胞标志物（α-平滑肌肌动蛋白（α-SMA），骨膜素（POSTN）），心肌细胞标记（Mhc6，Mhc7）和GAPDH。 </li></oL&gt; </li><li>培养小鼠血管内皮细胞: <ol><li>经过FACS分选和细胞收集（步骤4.3），离心细胞，在300×g离心5分钟。 </li><li>认真吸取掉上清液（排序缓冲区+媒体）并丢弃。 </li><li>重悬的GFP阳性细胞沉淀在1ml VEC媒体和在1ml的非内皮媒体的GFP阴性粒料。 </li><li>板每个样品的塑料腔滑的一个良好，成长，直到汇合。培养1周左右的GFP阴性细胞变得汇合和超过2周的GFP阳性细胞变得汇合（距离为3的样本中，成年小鼠）。改变媒体每隔两天。 </li></ol></li><li>免疫荧光染色: <ol><li>从细胞中除去介质并在4％多聚甲醛（PFA）进行30分钟，在室温固定。对于每次5分钟在磷酸盐缓冲盐水溶液（PBS）洗涤固定的细胞三次。 </li><li>从滑动和块，在5％牛血清白蛋白/ 1×PBS中除去腔孔1小时，在室温下。 </li><li>孵育幻灯片O 2 / N，在4℃用第一抗体（CD31，1:1,000或α-平滑肌肌动蛋白（α-SMA），1:100）。冲洗3×5分钟，在PBS中。 </li><li>稀释的二级抗体（Alexa的 - 氟568山羊抗鼠）1:在PBS 400中，添加到载玻片，并孵育1小时，在室温下。漂洗玻片3倍进行5分钟的PBS中。 </li><li>山中的Vectashield含DAPI孵育1小时，在4℃下观看之前。 </li></ol></li></ol>

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作者什么都没有透露。

在这里，我们描述了用于第一次的新方法从Tie2的-GFP小鼠胚胎和成年小鼠血管内皮细胞的分离。而此小鼠品系已被广泛用于内皮细胞群的分离，这是示出血管内皮细胞的选择性分离的第一份报告。由于VEC人口的脆弱性，我们已经开发出一种严格的协议，该协议允许对GFP阳性（与GFP阴性）细胞来自胚胎和成年小鼠的心脏瓣膜的单细胞分离。相比于使用整个胚或器官的Tie2-GFP小鼠14原出版，我们优化基于该脆弱的内皮细胞群的低丰度来分离细胞的选择人口胶原酶和清扫步骤。 VEC隔离以前只报道在有限的遗传和生物分子的工具大动物模型。这些工具以及在小鼠中建立，因此，在ABIlity分离小鼠血管内皮细胞可用于一组扩展的实验设计用于心脏瓣膜的研究。因此，这种方法的显著优点是血管内皮细胞可以暂时从野生型小鼠中，与瓣膜疾病和损伤的模型来分离。第二个优点是，血管内皮细胞可被分离并解剖后立即进行分析，防腐剂表达模式能够更准确地反映体内情况。另外，该隔离协议足以消除细胞培养数扩张和诱导的体外环境，因此潜在的表型变化被避免。 尽管新奇和本协议的实验效益，我们认识到，限制依然存在。首先，鼠阀内的血管内皮细胞种群的大小是非常小的，因此，需要以产生足够的RNA用于基因表达分析的多个定时胚胎胎。当THIs的使用多个育种加以克服，这可能对某些隔离后的分析工具的应用产生影响。这种限制一直是个挑战特别是为了进行VEC表型的更透彻的分析，包括分子谱和功能分析建立的GFP阳性的血管内皮细胞铺满培养物。因此，我们承认，不是所有的困难都被克服了我们的做法，但这种利益，我们正在努力解决的一个领域。 从瓣膜的区域隔离的血管内皮细胞的这种方法引入了从心内膜，或在心肌内的血管结构的铁GFP阳性，非瓣膜内皮细胞污染的可能性。迄今为止，血管内皮细胞的其它心脏内皮细胞群体的分子区别尚未确定。然而NFATC1基因的增强子区域已被确定并显示出特异性标记血管内皮细胞不经历EMT，并且没有其它的内皮细胞群的心脏18内。作为一种酶Cre模型（NFATC1 ENCRE）是可用的，未来的研究可以利用这一行的特殊性，以尽量减少污染风险。除了 ​​非瓣膜的Tie2-GFP-阳性细胞中，总有污染的，从在点肌细胞，其中所述瓣叶附着在室间隔和壁画心肌壁的环形区域的可能性。在此处未示出数据，我们最初开始研究，以从使用包被有抗-GFP和抗CD31抗体偶联的珠解剖心脏瓣膜区域隔离的血管内皮细胞。尽管这种方法是成功的用于分离的内皮细胞，我们经历显著细胞聚集来自相邻的非内皮细胞类型，并因此的VIC和心肌细胞污染我们的实验样品。这已被使用FACS分析参数已被设置为仅分离出单个细胞悬液避免s和PCR分析，以检测心肌细胞特异性基因表达已经控制了这一限制（ 图3）。而我们的污染可被视为极小，但仍可能避免在将来的双重选择GFP和内皮细胞特异性表面标记物的电位实验的复杂性。 使用这种协议中，我们有从胚胎和成年小鼠和如何使用此方法可用于RNA分离和GFP阳性和GFP阴性细胞群的细胞培养物提供了实例成功地分离血管内皮细胞。然而，这种方法并不局限于这些应用，并且可以被用于大量的分子，细胞和功能性的方法。此外，我们将Tie2-GFP的背景可以孕育与遗传小鼠模型，将允许在健康和疾病的VEC人群的比较研究。这种新颖的方法的发展将在第一时间，允许集中的研究研究血管内皮细胞的在阀门的开发和维护的贡献，并能揭示的内皮依赖性瓣膜病先前赏识的机制。

成熟的阀是由心脏瓣膜内皮细胞（血管内皮细胞）1的单层组成的专门的细胞外基质（ECM）穿插阀间质细胞（的VIC）和封装3分层的层。 ECM的作用是提供所有必要的生物力学特性的心动周期中，以承受在血流动力学的力常数的变化。阀门流脑在成人阀的营业额是紧紧的VIC这在很大程度上是静态和成纤维细胞样，在没有疾病的调节。除了​​VIC人口，心脏瓣膜内皮细胞（血管内皮细胞），形成一个不间断的内皮细胞上的阀尖2的表面上。而ECM和VICS的阀的结构和功能的重要性，已经描述了我们和其他人血管内皮细胞的作用不太熟知的。但是，这种比较小的细胞群体是对阀门的形成中胚临界和被描述为不正常的在阀病。 心脏瓣膜形成的胚胎开始时内皮细胞的房室通道和流出道区域内的一个子集进行内皮间质转化（EMT）和被称为心内膜垫1,3形式的肿胀。这些结构中的新转换的间充质细胞后分化成的VIC，形成了成熟的阀门。一旦EMT完成后，周围的咕内皮细胞，血管内皮细胞称为，形成防止损伤成熟阀不间断的内皮细胞层。此外，血管内皮细胞感测血流动力学环境和已被证明分子与底层的VIC通信以调节ECM动态平衡1,3。在病变瓣膜，血管内皮细胞的单层被破坏与细胞外基质的组织和异常变化改变了生物力学4,5协会。另外，在小鼠模型中的研究表明，血管内皮功能障碍是阀D的根本原因isease 1,6-9。作为血管内皮细胞中的阀门的开发，维护和疾病中发挥重要作用，这是重要的，我们完全定义，以推动该领域和了解疾病的机制，它们的时间的表型。 以前的工作由几个实验室已经成功分离出血管内皮细胞从猪和羊模型10-12。由于这些阀的尺寸大，单打通过擦拭和/或酶消化，接着通过多个不同的分离方法，包括磁珠细胞分离和单细胞克隆扩增一直有效，以产生纯的群体11-13。然而，这些模型可以限制因猪和羊的基因组限制了分子工具的可用性，除了成本高的不完整的注解。因此，在分离后的实验猪和羊的血管内皮细胞的可以是限制性的。小鼠模型是优选由于许多可能性遗传manipula灰和在胚胎和成年分子工具，但到目前为止，已经报道了在小动物模型没有VEC隔离。这可能是由于与小的组织样本，其中包括少数的细胞群，目前缺乏的血管内皮细胞特异性标记物的独特标识，从而防止基于抗体的分离方法工作的难度。 在本文中，我们报道了小鼠血管内皮细胞在胚胎和成年阶段，直接分离的新方法。这个协议利用的Tie2-GFP的小鼠，其表达GFP在所有内皮细胞类型，并已被广泛地用于研究内皮细胞群14。然而，目前的研究的新颖之处在于，这些小鼠中，在第一次已经被用来从阀隔离内皮细胞。通过小心地切开的瓣膜组织和一系列9酶消化随后通过FACS分选的，血管内皮细胞可被分离并用于各种实验技术，在cluding RNA提取和培养，直接下整理。

<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
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<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Alexa Fluor 568 Goat Anti-Rat IgG (H+L)</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:107px;">A-11077</td>
			<td style="width:148px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">70 &#956;m nylon cell strainer</td>
			<td style="width:107px;">BD Falcon</td>
			<td style="width:107px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">2.5% Trypsin</td>
			<td style="width:107px;">Invitrogen LT</td>
			<td style="width:107px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Bovine Serum Albumin (BSA), Fraction V, Heat Shock Treated</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:107px;">BP1600-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">CD31 rat anti-mouse antibody</td>
			<td style="width:107px;">BD Pharmingen</td>
			<td style="width:107px;">553370</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Chick Serum</td>
			<td style="width:107px;">Invitrogen LT</td>
			<td style="width:107px;">16110-082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Collagenase IV</td>
			<td style="width:107px;">Invitrogen LT</td>
			<td style="width:107px;">17104-019</td>
			<td>Prepared according to manufactuer&#39;s instructions</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">DNase I, RNase free (10,000 Units)</td>
			<td style="width:107px;">Roche</td>
			<td style="width:107px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">EBM-2</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:107px;">CC3156</td>
			<td>For VEC media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">EDTA, 0.5M Solution&#160;</td>
			<td style="width:107px;">Hoefer</td>
			<td style="width:107px;">03-500-506</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">EGM2 SingleQuot, Bulletkit</td>
			<td style="width:107px;">Lonza</td>
			<td style="width:107px;">CC-4176</td>
			<td>For VEC media</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">Fetal Bovine Serum (FBS), Heat Inactivated</td>
			<td style="width:107px;">Hyclone</td>
			<td style="width:107px;">SH3007103IH</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Hanks Balanced Salt Solution (HBSS)</td>
			<td style="width:107px;">Life Technologies</td>
			<td style="width:107px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Horse Serum</td>
			<td style="width:107px;">Invitrogen LT</td>
			<td style="width:107px;">16050-130</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Hybridization Oven</td>
			<td style="width:107px;">VWR</td>
			<td style="width:107px;">230301V</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Medium 199 1X</td>
			<td style="width:107px;">Corning Cell gro</td>
			<td style="width:107px;">10-060-CV</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Paraformaldehyde 16% Solution EM grade</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Pen/Strep</td>
			<td style="width:107px;">MP-Biomed</td>
			<td style="width:107px;">ICN1670049</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Permanox sterile chamber slide with cover</td>
			<td style="width:107px;">Thermo-Scientific</td>
			<td style="width:107px;">177429</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">Phosphate-Buffered Saline, 1X</td>
			<td style="width:107px;">Corning Cell gro</td>
			<td style="width:107px;">21-040-CV</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:115px;">PrimeTime Mini qPCR Assay</td>
			<td style="width:107px;">Integrated DNA Technologies</td>
			<td style="width:107px;">Acta2 (&#945;SMA), GAPDH, Myh6, Myh7, POSTN, CD31, vWF</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:115px;">StepOnePlus Real-Time PCR System</td>
			<td style="width:107px;">Applied Biosystems</td>
			<td style="width:107px;">4376600</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">SuperScript VILO Mastermix</td>
			<td style="width:107px;">Invitrogen LT</td>
			<td style="width:107px;">11755050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Tie2-GFP mice</td>
			<td style="width:107px;">Jackson Laboratories</td>
			<td style="width:107px;">3658</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">Tissue forceps #5 11cm</td>
			<td style="width:107px;">Dumont</td>
			<td style="width:107px;">14096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:115px;">Trizol</td>
			<td style="width:107px;">Ambion</td>
			<td style="width:107px;">15596018</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:115px;">&#945;-SMA anti-mouse antibody&#160;</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">A2547</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:115px;">VECTASHIELD HardSet Mounting Medium with DAPI</td>
			<td style="width:107px;">Vector Laboratories</td>
			<td style="width:107px;">H-1500</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

的Tie2-GFP细胞共定位与在胚胎和成年心脏瓣膜内皮细胞标记物。 为了确认的Tie2-GFP表达的血管内皮细胞来自胚胎和成年小鼠的特异性，免疫荧光法检测在从E14.5和3个月大的成年的Tie2-GFP制备组织切片的共定位与内皮细胞标记物，CD31此前公布的由我们实验室16使用方法小鼠。 如图1，血管内皮细胞共表达绿色荧光蛋白（ 图1，A，B，E，F）和CD31（ 图1 C，D，E，F）在成年（ 图1 B，D，F）和胚胎（ 图1中A，C，E）的阶段，因此，验证我们的模型进行后续的VEC隔离。 FACS分析鉴定在胚胎和成年心脏瓣膜从Tie2的-GFP的小鼠中分离的不同的GFP阳性和GFP阴性细胞群。 野生吨YPE C57 / BL6小鼠被用来设置GFP参数和单门控细胞的Tie2-GFP小鼠的约2％的同时显示胚胎和成年样品中的GFP阳性细胞显著富集通过FACS分析（ 图2）。根据图1中所示的共定位研究，这些细胞被认为是血管内皮细胞和产生平均61800总细胞（样品范围为39,000-77,000个细胞/样品）的成人样本中（N = 3），和8928细胞（8,015- 11000个/升）从E14.5胚胎的一个垃圾。 PCR分析证实了内皮细胞标记物中GFP阳性细胞和阀间质细胞标记物中GFP阴性细胞的Tie2-GFP的小鼠中分离的富集。 GFP阳性和阴性细胞群通过qPCR以下流式细胞仪的基因表达分析显示不同的分子轮廓。相对于绿色荧光蛋白阴性细胞群从的Tie2-GFP的胚胎和adul隔离TS，GFP阳性细胞富集的内皮细胞标记物CD31和vWF，（ 图3）的表达。此外，心肌细胞标记物（MYH6，MYH7）在这些细胞群体的表达非常低，这表明非内皮细胞的污染最小。与此相反，GFP阴性细胞从成人的Tie2-GFP小鼠和E14.5胚胎中分离出富集的阀间质细胞（VIC）标记物，α-SMA和骨膜素（POSTN）相对于GFP阳性细胞。这些标志物富集在绿色荧光蛋白阴性细胞分离自E14.5样品相比，成人高，由于成人的VIC的静态表型和活化标志物，因此低表达。 GFP阳性细胞的Tie2-GFP成年小鼠中分离，可以在体外培养。 的能力，以培养GFP阳性和GFP阴性细胞从成年样品中分离了检查基d于细胞形态学及免疫组化染色。使用先前由我们17说明我们显示在图4中的GFP阳性细胞两个星期培养（ 图4C）后出现圆形的形态（ 图4A）和共表达GFP的内皮细胞标记物CD31的协议。相比之下，非内皮细胞是阴性的GFP，九天后（ 图4D）显示间质样形态（ 图4B），并表示国际中心标记α-SMA。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51860/51860fig1highres.jpg" width="400" /> 图1中的Tie2-GFP-阳性细胞与CD31在胚胎和成年心脏瓣膜共定位。免疫荧光显示（Tie2-）GFP表达与内皮细胞标记物的关联（A，B），CD31（C，D）中在S二尖瓣在E14.5（A，C，E）和成人eptal传单（B，D，F）的阶段。该阀区域是在强调，C，E（E，F）合并的图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig1highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51860/51860fig2highres.jpg" width="500" /> 图2的Tie2-GFP阳性血管内皮细胞可通过流式细胞仪来识别。相较于同龄的C57 / BL6控制（A，C），从阀门的Tie2-GFP的胚胎（B）和成年人（D）中分离含有不同的GFP-阳性细胞群，如绿色表示。 GFP阴性的事件，显示为红色，收集作为对照。在（B）和（D）的数字表示GFP-p的平均％从事件的排序流式细胞仪的总数ositive细胞（N = 3）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig2highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51860/51860fig3highres.jpg" width="500" /> 图3 GFP阳性细胞富集为内皮细胞的标记物，而GFP阴性细胞表达与阀间质细胞相关的基因。的内皮细胞标记物（CD31，血管性血友病因子（vWF））和成人（MYH6）和胎儿代表的qPCR（ MYH7）的GFP阳性细胞E14.5（A）和成人（B瓣膜区隔离心肌标志物） 的Tie2-GFP的小鼠。音符的内皮细胞标记物和肌细胞相关基因非常低的水平的富集（C，D）折叠茶在阀门间质细胞标志物的表达NGESα-SMA从E14.5（C）和成人（D） 的Tie2-GFP的小鼠和POSTN孤立GFP阴性细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51860/51860fig3highres.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51860/51860fig4highres.jpg" width="500" /> 图4的Tie2-GFP阳性细胞保持在体外的内皮细胞标记物的表达。以下FACS，GFP阳性（A，C）和负（B，D）从成年小鼠的细胞群进行培养直到融合，可能会相衬成像（A，B）和荧光免疫染色（C，D）。 GFP阳性细胞后表达CD31（红）（℃）培养第10天。与此相反，没有在阴性细胞群体，其染色阳性的α-SMA，活化的VIC（D）的标记物检测的GFP表达。

Watch this Scientific Journal Video about Isolation of Murine Valve Endothelial Cells at JoVE.com

Isolation of Murine Valve Endothelial Cells

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-of-murine-valve-endothelial-cells

Nanyang Technological University

Research

JoVE Journal

Biology

14.2K 次观看.  The Ohio State University. 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.

视频: Isolation of Murine Valve Endothelial Cells

Isolation of Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

分离人脐静脉内皮细胞（HUVEC）

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

科研

生物学

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

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 ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

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 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 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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

鼠冠状动脉血管平滑肌细胞的分离

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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 Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

微型猪原主主动脉内皮细胞的隔离与培养

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.