这项研究由美国国家癌症研究所在美国国立卫生研究院院内研究计划的一部分得到了支持，并通过新华保险给予UA5CA152907。

1，收集条件培养基来测试潜在的血管生成<ol><li>成长伯或永生化细胞可以用于血管生成或抗血管生成潜力的天然的或低血清培养基中进行测试，并收集条件培养基。 <ol><li>或者，使用立即条件培养基，或等分并储存在-80℃下几个月。使用nonconditioned天然或低血清培养基作为阴性对照，并使用非条件完全生长培养基（10％FBS，或适当浓度）作为阳性对照。可替代地，测试血管生成的潜在刺激剂，补充剂木炭剥离介质缺乏生长因子与已知浓度的感兴趣的特定蛋白质，并比较单独的生长因子的培养基。 </li></ol></li></ol> 2，准备内皮细胞在测定前的<ol><li>两天来测定之前，通过3B-11细胞变成一个新的T-75瓶。细胞的传代次数是重要。该测定时效果最好的细胞是在第二和第六通道之间。该测定将无法正常工作，如果之前的第二或10次传代后的内皮细胞被使用。 </li><li>生长细胞24小时，在补充有10％FBS，2mM的L-谷氨酰胺，1mM丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>前一天测定，血清饥饿3B-11细胞中，如下所示： <ol><li>吸媒体从3B-11细胞。 </li><li> DMEM中添加降低血清培养基补充有0.2％FBS，2mM的L-谷氨酰胺，1mM的丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>生长细胞另外24小时。 </li></ol></li></ol> 3，试剂的配制在测定前注：BME浓度变化很大取决于很多。 BME不能很好地工作，如果浓度小于10毫克/毫升，所以BME制造商应购买，以确保现有接触收购一个足够浓很多。 <ol><li>除霜低生长因子BME在冰箱中。 BME温暖，当凝固，所以它必须保持低温才能使用。 BME能在冷藏条件下可保存数周，或分装冷冻在-80℃。 </li><li>减小的生长因子BME优于传统的生物医学工程。血管生成因子在BME缺乏会更容易地看到，因子从条件培养基对管形成的影响。 </li><li>要测试的解冻介质。 </li></ol> 4，准备内皮细胞前立即测定的<ol><li>洗3B-11细胞的DPBS。 <ol><li>如果可视荧光或共聚焦显微镜是期望的，洗涤3B-11细胞之前，钙黄绿素AM增加内皮细胞中的介质，以2微克/毫升的最终浓度。放置钙黄绿素标记的细胞在黑暗中，在37℃和5％CO 2之前测定的开始。溶解钙黄绿素的股票AM在DMSO中，并一次钙黄绿素稀释在DMSO中的媒体最终浓度应不超过0.1％。后从细胞30-45分钟洗净钙黄绿素AM（步骤4.1以上），并继续与实验如下。 </li></ol></li><li>加入1ml胰蛋白酶-EDTA至T-75烧瓶中Trypsinize细胞。 </li><li>胰蛋白酶消化完成后，中和胰蛋白酶通过再悬浮细胞至10ml的终体积在DMEM，10％FBS，2mM的L-谷氨酰胺，1mM的丙酮酸钠，100单位/ ml青霉素和100微克/毫升链霉素。 </li><li>通过100微米的细胞过滤过滤单元以去除团块。 </li><li>计数细胞，重悬细胞，以7.5×10 6个细胞/在DMEM毫升，10％FBS，2mM的L-谷氨酰胺，1mM丙酮酸钠，100单位/ ml青霉素的终浓度，和100微克/毫升链霉素。 </li></ol> 5，小管形成实验<ol><li>放置在冰箱或在冰上20-30分钟24孔板和1ml提示加载板，使得BME不PREM前aturely凝固。 </li><li>移液管加入250μl低生长因子BME成24孔板的每个孔中。避免未踩下吸管的最后一站，而配药产生气泡。注意使足够BME保持在中心孔的弯月面的形式时;如果矩阵是太薄，则细胞将只形成单层。 </li><li>孵育24孔板，在37℃和5％CO 2的30分钟以固化BME。 </li><li>一旦BME已成立，混合约300微升条件培养基用10微升悬浮3B-11细胞（约75,000细胞）。 <ol><li>调整的条件培养基和细胞取决于所用的内皮细胞类型镀的数量，并测试该条件培养基中的连续稀释液以证明活性。如果在不同条件下生长的比较单元，规范化条件培养基体积对细胞进行用于调节所述介质的数量。 </li></ol></li><li>管西港岛线升开始形成在2-4小时。根据在条件培养基中的血管生成因子的浓度，峰值管形成，可能会出现小时，并在测定的时间在3和12将需要被优化。如果用原代内皮细胞，而不是永生化细胞的工作，初始管形成可以通过几个小时延迟。 <ol><li>管经常会开始恶化内18小时，内皮细胞会进行细胞凋亡。 </li></ol></li><li>在高峰管形成时，从阱小心吸出介质，以避免破坏在BME管网络。 <ol><li>每1毫升的DPBS，然后吸出好洗。 <ol><li>立即可视化，使用连接到一台数码相机，或荧光/共聚焦显微镜，如果细胞用钙黄绿素AM如4.1.1倒置显微镜加入1 ml新鲜的DPBS到每个孔中，图像中的管网。 </li><li>要解决管网可视化后，加入4％多聚甲醛为每吸出，并培育15分钟，在室温下进行。 <ol><li>除去多聚甲醛固定，用DPBS冲洗两次，然后加入1ml新鲜的DPBS到每个孔中。 </li><li>显微镜下的图像之前固定的几个管会在此过程中断裂。 </li></ol></li></ol></li></ol></li></ol> 6，定量管网<ol><li>量化的管网中的几个不同的方式根据研究者的偏好，和几个计算机程序有能力来测量以下参数：管子的数目;中环/目数;的分支站点/节点数目;长管。 </li><li>使用有代表性的计算机程序，例如与血管生成分析器插件16，用于管网的定量的Image J。 </li></ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438, 932-936 (1038).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146, 873-887 (2011).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438, 937-945 (2005).</li><li>Bouis, D., Kusumanto, Y., Meijer, C., Mulder, N. H., Hospers, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+pro-+and+anti-angiogenic+factors+as+targets+of+clinical+intervention.">A review on pro- and anti-angiogenic factors as targets of clinical intervention.</a> Pharmacological research : the official journal of the Italian Pharmacological Society. 53, 89-103 (2006).</li><li>Ausprunk, D. H., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+proliferation+of+endothelial+cells+in+preformed+and+newly+formed+blood+vessels+during+tumor+angiogenesis.">Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis.</a> Microvascular research. 14, 53-65 (1977).</li><li>Chung, A. S., Lee, J., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tumour+vasculature:+insights+from+physiological+angiogenesis.">Targeting the tumour vasculature: insights from physiological angiogenesis.</a> Nature reviews. Cancer. 10, 505-514 (2010).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annual review of cell and developmental biology. 27, 563-584 (2011).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England journal of medicine. 358, 2039-2049 (2008).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England journal of medicine. 285, 1182-1186 (1056).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> The Journal of cell biology. 107, 1589-1598 (1988).</li><li>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12, 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature protocols. 5, 628-635 (2010).</li><li>Walter-Yohrling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+endothelial+cell+lines+as+models+of+tumor+endothelial+cells.">Murine endothelial cell lines as models of tumor endothelial cells.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 10, 2179-2189 (2004).</li><li>Connell, K. A., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+lymphoid+endothelial+cell+line+immortalized+by+simian+virus+40+binds+lymphocytes+and+retains+functional+characteristics+of+normal+endothelial+cells.">A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.</a> Journal of immunology. , 144-521 (1950).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+contribution:+Angiogenesis+Analyzer.">ImageJ contribution: Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li></ol>

作者什么都没有透露。

血管生成是涉及在生理和病理过程。研究参与血管生成的机制要求使用概括在血管生​​成中的重要步骤测定的。内皮细胞管形成测定提供了几个优点优于其它试验。很容易设置，是比较廉价，生产小时内小管，并且是可量化的。此外，它可以在24位或96孔板内完成，因此可用于高通量筛选，以确定该刺激因子或抑制血管生成。虽然我们用3B-11小鼠内皮细胞为我们的实验中，测定可与各种人或鼠内?...

血管生成，新血管从先前存在的血管的发展，是各种过程，包括器官发育，胚胎发育和伤口愈合1-3至关重要。新开发的血管，内皮细胞，供应氧气和营养物质，内衬以组织，促进造血细胞的免疫监视和清除废物的2,4。血管生成是至关重要的胚胎和胎儿发育过程中。然而，这种方法保持休眠期间除外伤口愈合，骨生长，怀孕次数或月经周期1-3期间在成体中。 在过去的二十年中，调节血管生成的关键分子机制已经开始出现。血管新生是一个严格监管的情况下，通过亲和抗血管生成信号，包括整合，趋化因子，血管生成素，氧传感代理商，交界性分子和内源性抑制剂5平衡。一旦proangiogenic信号，如碱性成纤维细胞生长因子（bFGF），血管内皮生长因子（VEGF），血小板衍生生长因子（PDGF），和表皮生长因子（EGF），激活内皮细胞受体的内皮细胞释放的蛋白酶降解基底膜。内皮细胞，然后增殖和迁移，以每6,7天数毫米的速度形成豆芽。 血管生成与多种病理状况，包括癌症，牛皮癣，糖尿病性视网膜病，关节炎，哮喘，自身免疫性疾病，感染性疾病，动脉粥样硬化和8-10相关联。由于血管生成的各种疾病的重要性，了解调节这一过程是更好的治疗设计的关键基因和途径。 在管道形成试验是一种快速和定量的方法，用于确定参与血管生成基因或通路。于1988年首先描述，此测定法的基本原则是内皮细胞保持分裂和迅速地响应于血管生成的信号11-13迁移的能力。此外，血管内皮细胞被诱导分化和形成管样结构，当上的基底膜提取物（BME）的基质中培养。这些管道包括通过连接复合体连在一起的内皮细胞围成的管腔。管形成很快发生最管在该测定中形成内2-6小时，这取决于数量和血管生成的刺激的类型。 几种类型的内皮细胞可用于该测定中既包括原代细胞和永生化细胞系14,15。本文使用的细胞系是小鼠3B-11，但是相同的方法可以与其他内皮细胞系如SVEC4-10（小鼠）或原代内皮细胞，例如血管内皮细胞（人）的细胞被应用。这取决于细胞系用于与是否内皮策LLS转化或未转化，优化将需要被进行，以确定所需要的适当的管道形成了理想的时间。

<table border="0" cellpadding="0" cellspacing="0" width="651">
	<tbody>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:339px;">Item</td>
			<td rowspan="2" style="width:189px;">Manufacturer</td>
			<td rowspan="2" style="width:124px;">Catalog #</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="93">
			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="169">
			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="55">
			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25300-054</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="74">
			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">14190-144</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Fetal Bovine Serum</td>
			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
		</tr>
	</tbody>
</table>

endothelial cell tube formation assay for the in vitro study of angiogenesis

血管生成是用于正常组织发育和伤口愈合的重要方法，而且还与多种病理状况相关联。通过此协议，血管生成可在体外快速的，可量化的方式进行测量。初级或永生化的内皮细胞被混合的条件培养基并接种于基底膜基质。内皮细胞形成毛细血管样结构响应于在条件培养基中发现的血管生成信号。该管形成迅速发生与血管内皮细胞开始1小时内和含腔，小管开始出现在2个小时的赞同。管可以使用相衬倒置显微镜进行可视化，或者细胞可以与现有的测定法和管通过荧光或共聚焦显微镜观察钙黄绿素AM被处理。的分支站点/节点，回路的数量/网格，或数字或形成管的长度可以很容易地量化为在VITR措施Ø血管生成。总之，本试验可用于确定基因和参与了促进或抑制血管生成中具有快速，可重复的，和定量的方式通路。

鼠标3B-11内皮细胞接种在固化低生长因子BME - 在该试验中，产品的基质胶，使用 - 和随后随着时间的推移。 如图1中 ，由任一鼠角质细胞或成纤维细胞分泌的血管生成因子能够诱导管的形成随时间的。血管内皮细胞迁移，并开始在1-2小时电镀形成小分支。最大管形成是通过使用先前从角质细胞获得的条件培养基4-6小时到达。 24小时，一些分支机构仍然存在，但许多细胞凋亡成为和管?...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

小管形成实验是测量在体外血管生成一种快速的，可量化的方法。内皮细胞结合的条件培养基并接种于基底膜提取物。管形成时小时内新形成的小管很容易量化。

内皮细胞小管形成实验的<em&gt;在体外</em&gt;研究血管生成

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

endothelial-cell-tube-formation-assay-for-vitro-study

Research

JoVE Journal

Biology

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

59.0K 次观看.  American University. 小管形成实验是测量在体外血管生成一种快速的，可量化的方法。内皮细胞结合的条件培养基并接种于基底膜提取物。管形成时小时内新形成的小管很容易量化。 

视频: Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

科研

生物学

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

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, branching, 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 within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an 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. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

优化的纤维蛋白凝胶珠检测血管生成的研究

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

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

基于一个基质胶管形成实验，以评估对肿瘤细胞的血管生成活性

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化：调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

内皮细胞粘附性定量<em&gt;在体外</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

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 ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.