SAS and MGA are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. MMH has support from a Peter MacCallum Foundation Grant.

IL-3和制备的源WEHI-3D​​的空调中等注意:小鼠粒细胞白血病细胞系WEHI-3D​​是培养以产生含IL-3条件培养基。 <ol><li>培养WEHI-3D​​中的Dulbecco改进的Eagle培养基（DMEM），10％胎牛血清（FBS），1％的长寿命补充谷氨酰胺，50μg/ ml的庆大霉素。接种5×10 6个细胞中生长的对数相到50ml在一个T175 cm 3的组织培养瓶的新鲜培养基并生长约7天，或者直到细胞已经由约24通过增长其数期- 48小时（避免过度培养细胞死亡）。条件培养基是能够被存储为多个年在-20℃，所以200批次 - 400毫升能在同一时间产生。 </li><li>滗析从烧瓶和旋转流体和细胞在1,000×g下15分钟以除去细胞和细胞碎片。拆下顶部90％supern的atant。通过0.22微米的过滤器单元进行过滤。 </li><li>等分试样WEHI-3D​​的条件培养基（CM）在1ml 50毫升，并在-20℃下200毫升卷进行存储或-70℃。小等分试样用于测定制备，用于制备培养基为因子依赖性细胞系的通道中间的卷，以及用于长期储存的卷是有用的。 IL-3是在4℃下，如果在无菌条件下使用，所以解冻介质可以存储几个星期相对稳定。 </li><li>另外，使用重组小鼠IL-3的稀释于培养基中，通过0.22微米的过滤器单元过滤后50ng / ml的水平。 </li></ol> 2.培养和评价的VEGFR-2-EpoR的-的Ba / F3和控制的Ba / F3细胞系的<ol><li>培养控制Ba / F3细胞在DMEM，10％FBS，50μg/ ml的庆大霉素或青霉素/链霉素补充，1％稳定的L-谷氨酰胺和10％WEHI-3D​​-CM。有关从细胞每三天传代细胞以1:15稀释在对数期生长。 </li><li>培养VEGFR2-EpoR的-Ba / F3细胞在DMEM，10％FBS，50μg/ ml的庆大霉素，1％稳定的L-谷氨酰胺和10％WEHI-3D​​-CM和1毫克/毫升G418。传代细胞1:15稀释在对数期生长的细胞。有关嵌合受体的构建和表达的更多详细信息，请参阅24。 </li><li>收获控制的Ba / F3或VEGFR2-EpoR的-Ba / F3细胞中旬对数期的文化。轻轻吸取从烧瓶底部除去这些非贴壁细胞。在小鼠张力磷酸盐缓冲盐水，pH值7.3（PBS）中洗涤三次，（10毫升，离心，在750×g下5分钟以回收细胞沉淀）以除去含IL-3培养基。 </li><li>洗涤细胞用DMEM和添加剂一次，而不WEHI-3D-CM或重组IL-3，然后重悬在该培养基中以7.4×10 4个细胞/ ml（即每13.5微升1,000个细胞的浓度; 10,000个细胞每135微升通过使用血细胞计数器计数测定）为在分别测定的半定量或定量的版本使用。 </li><li>评估由台盼蓝排斥（警告）的活力细胞。混台盼蓝的PBS（0.4％）1:1的细胞群，并在血细胞计数器计数的最低100个细胞。这样会占用染料细胞被认为是死亡或濒临死亡。的大于98％的生存力是需要进行测定。 </li></ol> 3.半定量分析<ol><li>添加包含在13.5微升IL-3缺陷型培养基中以在RT 72孔微孔板的孔中洗涤细胞（1000细胞）。小心aliquotting确保细胞沉淀在重力作用下不偏细胞浓度时混合细胞悬液。使用以及校准P20自动化移液器和蒸压提示。 </li><li>添加测试样品和对照的孔中在10％体积（1.5微升，使得每孔含有1,000个细胞15微升的最终体积）使用校准P20移液管或，优选，P2移液管。以CA重，以确保样品在pH值，盐和其他可能的细胞毒性/细胞抑制剂的物质而言的培养条件为Ba / F3细胞兼容。如果可能的话，使用兼容的介质或缓冲液（如 DMEM或PBS，分别），或在此类介质或缓冲液稀释。研磨用相同的尖端将确保混合。 </li><li>对于对照样品，包括（i）中单独的含无IL-3; （ⅱ）WEHI-3BD-CM加入到培养基单独以10％的最终体积和/或（iii）VEGF-A稀释至在单独介质100纳克/毫升，不含有IL-3。 </li><li>填用无菌水，PBS或培养基的微孔板的任何未使用的孔中，放置的潮湿容器中的板（与水浸泡棉纸）允许气体交换。孵育在容器内的细胞在10％的CO 2的潮湿气氛中。该测定可以舒适地设置在3小时由一个人如果测试样品和媒体组件是可用的。 </li><li>评估板通常后16小时在培养其阳性和阴性样品之间的时间区别是显而易见的。参见图3的细胞如何出现在文化的例子。 100×放大率 - 使用标准倒置相差显微镜在40分析平板。 注意:包含韦尔斯支持生长因子如IL-3或VEGF-A将含有呈圆形和半透明的细胞。而不支持生长因子或与单独的培养基孔中会具有降低的圆形细胞，以及死亡或正在死亡的细胞和细胞碎片（例如， 图3C）的号码。在24-48小时后孵化评估法是最佳的灵敏度。 </li></ol> 4.定量生物测定<ol><li>在IL-3缺陷型培养基中添加洗涤细胞（10,000个细胞在135微升），以在RT标准的96孔微量滴定板的孔中。小心aliquotting以确保重力不影响CE细胞沉淀过程中，混合细胞悬液LL浓度。 </li><li>添加测试样品和对照到孔在使用校准吸管（P20）10％体积（15微升）。小心确保样品与pH值，盐或其他潜在的细胞毒性/细胞物质方面的培养条​​件为Ba / F3细胞兼容。如果可能的话，使用兼容的介质或缓冲液（ 例如 ，DMEM或PBS），或在此类介质或缓冲液稀释。过滤器采用0.22微米孔径的醋酸纤维素离心管过滤装置灭菌小容量。 </li><li>孵育细胞，生长因子和/或抑制剂剂的混合物中的10％的CO 2的潮湿气氛48小时。具有水渍薄纸，并允许气体交换的潮湿容器中进行测定。在温育期结束时，评估使用的，低于该列出的替代性方法中的一种测定是在井的活细胞数目的替代标志。 </li><li> 替代＃1:3 H-thymidiNE公司注册 注意:此量化是专为96孔板形式。 <ol><li>在37℃下（每孔150μl的体积）为48小时测定板的温育后，在50微升测定培养基的体积添加3 H-胸苷（的Ba / F3培养基没有IL-3 / WEHI-3D ​​CM）每孔以20微居里/ ml的浓度，得到的每孔1微居里的最终剂量。 </li><li>孵育板用于使用细胞收集器收获细胞前在37℃下再进行4小时。通过将过滤器在闪烁体提取各个样品，并用液体闪烁计数器定量。 </li></ol></li><li> 替代＃2:细胞内ATP生物发光检测 注意:此方法使用当与细胞裂解物相结合，可产生反映在人口中的活细胞的生物发光信号的ATP的监测试剂。 <ol><li>裂解细胞以提取ATP和使用ATP监测试剂，以产生一个根据制造商的说明发光信号。使用带有一个96孔板格式兼容光度计读取发光。 </li></ol></li><li> 替代＃3:由活细胞的指示剂染料酶催化还原 注:基于刃天青试验显示良好的相关性细胞活力。 <ol><li>结合培养细胞用刃天青染料和量化使用根据制造商的说明在荧光读板器测定。 </li></ol></li></ol>

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史蒂芬A.堆和Marc M.亚琛是股东Circadian技术有限公司，公司通过瞄准生长因子VEGF家族发展疗法。

此处所描述的测定法依赖于使用高存活率的细胞，这是依赖于生长因子。因此细胞需要仔细培养，以确保它们是因子依赖性的，并保留嵌合受体的表达。确保介质新鲜的，而不是存放过长的时间，并且WEHI-3D​​ CM为高活性是重要的。细胞需要从含培养基的IL-3彻底洗涤进测定培养基，以确保无残留的IL-3将细胞暴露于配位体抢救时污染测定。作为配体可以来以多种不同的形式，护理需要用于测定制备这些时要采取。防腐剂，抗菌剂，盐或不同pH的缓冲液可影响测定中，这就是为什么未转染的Ba / F3细胞的平行测试可以告知缓冲器相关的问题。 我们同时使用半定量和定量协议。半quantitati已经测定允许对样品的活性的快速评估提交到完全定量测定法，其需要更多的时间和样品之前。出版生成数据时定量方法中使用。用于检测培养物的生存力的可供选择的方法是相对简单的。的定量测定已经适应许多不同的格式增殖的定量，并且我们已经使用（3-（4,5-二甲基吡啶-2-基）-2,5-二苯基溴化） MTT 30，细胞ATP（未公布的），基于刃天青染料（ 图2）中 ，用成功3 H-胸苷31的生物发光检测。近年来，我们从使用辐射，以适应生物发光方式演变路程，已经使用，由于其成本效益的基于刃天青染料。所有办法给予适当的结果和选择取决于试剂的可用性和一个在给定的实验室/机构或熟悉特定的方法ssociated专用板的读者。 故障排除围绕，i）的IL-3的CM，ⅱ）VEGFR-2-EpoR的-Ba / F3细胞和iii）刺激配体测定的三个主要组成部分的活性。 IL-3中的CM由于WEHI三维细胞不足在初始培养，差的储存条件，过度冻融循环或使用在太高的稀释度可以创建不良生长培养，将阻碍该测定差水平。这通常是在对照孔检测为细胞群体越来越慢，失去正常的亮又圆的外观。的VEGFR-2-EpoR的-的Ba / F3测定细胞可随着时间的推移，如果保持在培养长时间失去的嵌合构建体的细胞表面表达。保持液态氮的股票不错的数据使细胞的新鲜培养高度表达嵌合受体是随时可用。如果需要高表达群体可以是通过在VEGFR-2的配体生长的细胞，并选择人口表达由流动的高水平的嵌合受体的流式细胞术分选来实现。然后将这些细胞进一步培养扩增冻结。 而该技术是用于与VEGFR-2的胞外结构域分析相互作用的便利和简化的替代它不能被看到，以取代在那里初级内皮细胞天然受体在其他VEGFRs，共受体如存在下探测更复杂的实验neuropilins。在其上该测定是基于细胞（B细胞）是从血管内皮细胞不同，因此不应被用来研究在血管内皮细胞的VEGFR-2的激活作出反应的细胞质信号传导途径和转录因子。然而，该方法提供了以分析受体 - 配体相互作用的有用方法，在于该检查与固定化雷杰普的配体的相互作用的简单的结合测定之间器构造（例如可溶性受体细胞外区或融合到免疫球蛋白的受体胞外区域），和受体结合分析和结合试验中的初级内皮细胞可见下游效应。它提供了一个系统，以允许受体的结合和交联，前两个阶段由许多受体型酪氨酸激酶通常引发它们的信号转导级联。其在许多学术和商业设置应用已经显示出评估变体的配体或受体 - 配体相互作用的抑制剂特别是当它被接受为用于检查结合特征的有用试验。 该测定也是的受体胞外域或其配体的新的抑制剂的分析是有用的。该测定可以进一步用于迅速筛选基因组的突变的情况下看到的配体或受体胞外区的变体（包括继承和体细胞）在人戈发现在疾病如癌症32,33内斯以确定它们的功能性后果。因此本VEGFR-2测定的应用很可能在未来的扩展。此外，该测定的基本的一般方法，可以更广泛地用于在细胞表面的受体酪氨酸激酶，它们构成一个大的和重要的家庭信令在健康和疾病的分子的使用。

血管内皮生长因子（VEGF）家族的分泌蛋白的生长因子和它们的同源细胞表面受体是可溶性配体和膜包埋的受体，分别在穿过细胞膜转导信号该函数的一个重要和多样的基团。它们主要发挥作用的内皮细胞，而且在和上皮来源的细胞的那些免疫系统1,2。信令由配体激活的VEGF受体（VEGFRs）是在主要的病状，如年龄相关性黄斑变性和癌症关键的，和治疗啮合途径靶向它们是在常见的临床使用（例如，单克隆抗体贝伐单抗靶向的VEGF-A）的3,4。 一项所述的VEGF家族的复杂性是存在于自然界（可溶性配体VEGF-A，VEGF-B，VEGF-C，VEGF-D，由parapox病毒家族ORF和蛇毒VEGF，加上其他编码的VEGF蛋白质的多样性抑制VEGF-A）2亚型。 这些配体与受体酪氨酸激酶家族的三个成员，即VEGFR-1，VEGFR-2和VEGFR-3相互作用。这些受体可变地表达于不同的细胞类型，但该行的所有大小5的血液和淋巴管内皮细胞的表面上常常共表达。 VEGFR-2可以结合哺乳动物配体VEGF-A 6，VEGF-C 7和VEGF-D -8,9-以及口疮病毒VEGF 10和蛇毒VEGF的11。 VEGFR-2在驱动血管发生在胚胎发育（新血管从预先存在的血管的生长），伤口愈合，癌症和眼病主要作用。在这些情况下，配体例如VEGF-A，-C和-D结合并激活血液血管内皮细胞12-15受体。上淋巴管内皮细胞，VEGFR-2在淋巴管生成的作用，新的淋巴管16的形成。 VEGFR-2还可以促进扩张和主要动脉和淋巴管在健康组织和疾病17扩张。 VEGFR-2的完整的理解:配体的相互作用是因此对于抑制剂的开发用于治疗血管生成依赖性疾病18重要。而VEGF-A结合的对VEGFR-2的大部分同种型，是必需的VEGF-C和VEGF-D的蛋白水解裂解，以释放由表现出高亲和力结合VEGFR-2 19,20的VEGF-同源结构域的片段。 我们已经开发出一种生物测定来监测，其被设计来规避初级内皮细胞，这是技术上难以通过，购买昂贵的和培养的需要VEGFR-2的配体（需要专门介质）21和表达多种VEGFRs和相关的共受体22。旨在螺柱当与其他VEGF受体或共受体VEGFR-2的异源可引起不希望的复杂性ý二进制受体-配体相互作用，评估活性归属于特定的受体，或评估抑制试剂的效果。23。生物测定保留在细胞膜相关受体的流动性，并允许的配位体的结合能力并交联的VEGFR-2的细胞外区域的评价。 生物测定依赖于其中（在这种情况下，VEGFR-2）VEGF受体的胞外区融合至跨膜和促红细胞生成素受体（EpoR的），细胞因子受体家族的一个成员的胞内区域的创建的嵌合受体8,24。此融合蛋白然后在从属因子原B细胞系的Ba / F3表示，在其上的刺激与能结合和交联的受体的胞外结构域导致胞质效应区，其能够激活一个配位体通过JAK激酶（JAKS）转导存活信号，促进细胞的存活和/或增殖。与此相反，全长VEGFR-2在相同的细胞类型，并刺激配体表达，不促进细胞存活和增殖，表明VEGFR-2通路的近端信令效应不在此细胞类型可用。 我们已经使用该测定在各种情况下，探索新的VEGFR-2的配体10,19,20,24-29的结合。在与VEGFR-3-EpoR的-的Ba / F3测定结合，我们比较了VEGF-C的结合和交联的VEGFR-2和VEGFR-3 30的相对活性和VEGF-D的生长因子。该测定法已被用于表征中和单克隆抗体对VEGFR-2或VEGF-D，可溶性VEGFR-2捕集和拟肽靶向VEGF家族31的抑制活性。该试验也用于显示的VEGF的不同ORF病毒株结合交联的VEGFR-2在测试之前在初级内皮细胞的能力，并<s达&gt; 10,26。该测定是对的VEGF的突变体的快速筛选，可以它们被引入到更费力内皮细胞分析25，或当用于净化生长评估协议因子27前被快速评估活动特别有用。 我们描述了检测容易进行，并且半定量版本允许监测的生长因子，抗体或其他实验的可溶性受体结构域的生产或纯化时有时需要快速测定。使用该测定的易用性使得它与从血液或淋巴管衍生自特定的组织或器官系统主内皮细胞进行进一步和更完全的研究，一个理想的补充。

<table border="0" cellpadding="0" cellspacing="0" width="1110">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Trypan Blue</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:181px;">T8154</td>
			<td style="width:424px;">0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 Sulphate (Geneticin)</td>
			<td>Invivogen</td>
			<td>ant-gn-5</td>
			<td>Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3H-Thymidine</td>
			<td style="width:183px;">PerkinElmer</td>
			<td style="width:181px;">NET-027</td>
			<td>This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vialight Plus Kit</td>
			<td>Lonza</td>
			<td>LT07-221</td>
			<td>Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prestoblue Cell Viability Reagent</td>
			<td>Invitrogen</td>
			<td>A13261</td>
			<td>Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Nunc Minitray with Nunclon Delta Surface (72 well)</td>
			<td>Thermo Scientific</td>
			<td>136528</td>
			<td>Small microtitre plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">96 well Tissue Culture Plate</td>
			<td style="width:183px;">Falcon, Corning Inc.</td>
			<td style="width:181px;">353072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">DMEM (1X)</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">11965-92</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">GlutaMAX (100X)</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Foetal Bovine Serum</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">&#160;10099-141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Harvester</td>
			<td style="width:183px;">Tomtec Life Sciences</td>
			<td style="width:181px;">N/A</td>
			<td>Tomtec Harvester, 96 Mach 3M Cell Harvester</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Scintillation Counter</td>
			<td style="width:183px;">LKB Wallac</td>
			<td style="width:181px;">1205</td>
			<td>LKB Wallac 1205 Betaplate Scintillation Counter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">UniFilter-96 GF/B</td>
			<td style="width:183px;">Perkin Elmer</td>
			<td>6005177</td>
			<td>White 96-well Barex Microplate with GF/B filterof 1 &#181;m poresize</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Gentamicin</td>
			<td style="width:183px;">Gibco, Life Technologies</td>
			<td style="width:181px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td style="width:183px;">Gibco, Life Technologies</td>
			<td style="width:181px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:323px;">0.22um pore cellulose acetate centrifuge tube filter unit</td>
			<td style="width:183px;">Costar, Corning Inc.</td>
			<td style="width:181px;">8160</td>
			<td>Centrifuge tube filters have a 0.22&#181;m pore CA membrane-containing filter unit within a 500&#181;l capacity polypropylene microcentrifuge tube.</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Fluorescence Reader</td>
			<td style="width:183px;">BioTek</td>
			<td style="width:181px;">N/A</td>
			<td>BioTek Synergy 4 Hybrid Microplate Reader&#160;</td>
		</tr>
	</tbody>
</table>

a simple bioassay for the evaluation of vascular endothelial growth factors

参与血管生物学受体酪氨酸激酶及其相互作用的配体的分析常常是具有挑战性的，由于相关的受体，范围广泛的相关配体和处理专门内皮细胞的原代培养物的难度家庭的组成型表达。这里，我们描述用于检测配体的血管内皮生长因子受体-2（VEGFR-2）中，促进血管生成和淋巴管生成的信号的键传感器生物测定。编码VEGFR-2的胞外（配体结合）区的融合与跨膜和促红细胞生成素受体（EpoR的）的细胞质区域的cDNA，在因子相关性细胞系的Ba / F3表示。该细胞系生长在白细胞介素-3（IL-3）和该因子的结果的撤出在细胞的死亡存在24小时之内。在VEGFR-2 / EpoR的受体融合的表达提供了一种替代机制，促进生存和势的在能够结合和交联的融合蛋白（ 即 ，一个可以交联的VEGFR-2的胞外区）的胞外部分的配位体的存在下稳定转染的Ba / F3细胞的LLY增殖。半定量的方法，其中配位体和细胞小体积在24小时允许快速结果，并且涉及活细胞数目的替代标志物的定量方法:在测定可以通过两种方式来执行。该测定是比较容易进行，是高度响应于已知VEGFR-2的配体，可容纳的VEGFR-2的信号，如单克隆抗体的受体或配体，和可溶性配体陷阱胞抑制剂。

在这一节中，我们显示的实验展示一VEGFR-2-EpoR的-的Ba / F3生物测定（参见图1用于测定的原理）的基本特征的结果。其他已发表 ​​的研究表明在测定的替代VEGFR-2的配体，突变的VEGF分子和抑制性单克隆抗体8,10,19,24-30的更广泛的应用。 此处呈现的数据表示，其中的VEGFR-2-EpoR的-的Ba / F3生物测定来量化三种重组人配体（VEGF-A 165，VEGF-C和VEGF-D）具有特异性VEGFR活性的测定-2的抑制性单克隆抗体（贝伐单抗）的存在下的VEGF-A 165的数据已被归一化相对于响应于VEGF-A 165单独（ 图2）。正如预期的那样，贝伐单抗阻断VEGF-A的测定中165的作用，但对VEGF-C和VEGF-D的活性没有影响。 在半定量版本测定的或者其发展过程中观察测定时，观看使用标准倒置相差显微镜可以提供关于该测定的进度有价值的信息的检测。的测定（ 图3）的分析表明，当用含有IL-3的培养基，或配体孵育VEGFR-2， 例如，VEGF-A的VEGFR-2-EpoR的-的Ba / F3生物测定细胞的良好的可行性。这些细胞是大和半透明的，并且有粒度的在培养液中的细胞的细胞质或细胞碎片没有或最小的证据。在阱，没有额外的生长因子已经有减少的细胞数目和少数健康细胞的证据。目前细胞有显著粒度在他们的细胞质和细胞碎片是文化的一贯特点。 48小时后没有存活的细胞的迹象。 图1原理图描述了VEGFR-2-EpoR的-的Ba / F3生物测定的原理。（A）中的Ba / F3细胞因子依赖性和需要的外源生长因子如IL-3通过对刺激细胞生长/活力内源性的IL-3受体和JAK通路的影响。如果EpoR的是在这些细胞中拯救表达也可以通过JAK途径发生。全长受体酪氨酸激酶的表达，如VEGFR-2的结果中最小的信令作为VEGFR-2激活的下游目标不接合JAK通路。与稠合的EpoR的跨膜和胞质区的VEGFR-2的胞外区的嵌合受体，当转染到Ba / F3细胞和刺激的特定VEGFR-2的配体，能够激活JAK路径和驱动单元的存活和增殖。 （B）的VEGFR-2-EpoR的-Ba / F3细胞表达嵌合VEGFR-2-EpoR的受体，可以使用流式细胞术进行定量的显著水平。一旦洗出含有培养基的IL-3的细胞置于各种哪一个驱动器单元的任一存活/增殖的培养条件。未转染的Ba / F3细胞或VEGFR-2表达EpoR-Ba / F3细胞没有特殊的生长因子无法生长/生存。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53867/53867fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53867/53867fig2.jpg" /> 图2. VEGFR-2的配体中的VEGFR-2-EpoR的-的Ba / F3生物活性测定。IL-3依赖性Ba / F3细胞表达小鼠VEGFR-2-EpoR的嵌合体的评价在培养基中接种无IL-3（除对于的+ IL-3的子集，所指示的）加人VEGF-A 165，VEGF-CΔNΔC（VC）（这是VEGF-C的形式，它由中央区域和不包括N和C末端前肽），或VEGF-DΔNΔC（这是VEGF-D的形式，其中包括中央区域和不包括N和C末端前肽（VD））在100纳克/毫升和人源化中和性单克隆抗体贝伐单抗（其结合并抑制VEGF-A，但不是VEGF-C或VEGF-D），或控制非中和抗体曲妥单抗，如在0.75微米表示。不含有另外的生长因子NF =网上平台。四十八小时后，相对的活细胞数，用细胞ATP的生物发光检测定量。竖线代表机构（归一化与应对VEGF-A 165独自一人，第一棒）和三个独立的实验平均值的标准误差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53867/53867fig2large.jpg" target="_blank">请点击此处到v</a>IEW这个数字的放大版本。 <img alt="图3" src="/files/ftp_upload/53867/53867fig3.jpg" /> 图3. VEGFR-2-EpoR的-Ba / F3细胞是对生存的外部生长因子依赖性。VEGFR-2-EpoR的-的Ba / F3生物测定的实例，其中的IL-3依赖性Ba / F3细胞表达小鼠VEGFR- 2-EpoR的嵌合体在含10％WEHI-3D ​​的条件培养基，它提供的IL-3，（B）的100毫微克/ ml人VEGF-A 165（无IL-3），或（C）用培养基中（A）的接种没有IL-3或其他生长因子。培养中描绘24小时到试验示出了IL-3（A）或VEGF-A 165（B）的存在下高度的活细胞，而在没有IL-3或另外的生长因子（C）的细胞死亡的培养和红uced可行性是显而易见的。秤酒吧100微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53867/53867fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

我们描述了检测，定量和监测血管内皮生长因子家族的配体的成员的活性的简单的基于细胞的生物测定法。该测定法使用在因子相关性细胞系中表达的嵌合受体的配体，以提供受体结合和交联的半定量或定量的评估。

一个简单的生物测定血管内皮生长因子的评价

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

a-simple-bioassay-for-evaluation-vascular-endothelial-growth

Nanyang Technological University

Research

JoVE Journal

Biology

9.6K 次观看.  Peter MacCallum Cancer Centre. 我们描述了检测，定量和监测血管内皮生长因子家族的配体的成员的活性的简单的基于细胞的生物测定法。该测定法使用在因子相关性细胞系中表达的嵌合受体的配体，以提供受体结合和交联的半定量或定量的评估。 

视频: A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

科研

生物学

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

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 ...

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 ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

使用 En 面免疫荧光染色直接观察血管内皮细胞

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic nephropathy, are associated with VEGF dysregulation in the kidney. The factors that govern VEGF under physiologic conditions in the kidney are not well-understood. Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs. Given that LH receptors are found in the kidney, we, at Zietchick Research Institute, hypothesized here that LH also helps regulate VEGF expression in the kidney as well. To provide evidence, we aimed to show that LH levels are able to predict VEGF levels in the mammalian kidney. Most VEGF-related investigations involving the kidney have used lower order mammals as models (i.e., rodents and rabbits). To translate this work to the human body, it was decided to examine the relationship between VEGF and LH in higher order mammals (i.e., bovine and porcine models). This protocol uses the total protein lysate from the kidney cortex. Keys to this method&#39;s success include procurement of kidneys from slaughterhouse animals immediately after death as well as normalization of analyte levels (in the kidney extract) by total protein. This study successfully demonstrates a significant linear relationship between LH and VEGF in both bovine and porcine kidneys. The results are reproducible in two different species. The study provides supporting evidence that the use of kidney extracts from cows and pigs are an excellent, economical, and abundant resource for the study of renal physiology, particularly for examining the correlation between VEGF and other analytes.

Presented here is a protocol for utilizing a cortical kidney extract preparation and total protein normalization to demonstrate the correlation between vascular endothelial growth factor and luteinizing hormone in the mammalian kidney.

在肾脏皮质提取物中演示血管内皮生长因子与叶黄素激素之间的线性关系

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

分离人原代瓣膜细胞进行体外疾病建模

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...