Name: optimized fibrin gel bead assay for the study of angiogenesis
Uploaded: 2007-04-29T00:00:00
Description: Watch this Scientific Journal Video about Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis at JoVE.com

<p class="jove_step">制备细胞</p><ol><li> M199/10％胎牛血清/笔链球菌（1:100）前1-2天钉珠人脐静脉内皮细胞和成纤维细胞。 </li><li>开关中EGM - 2（Clonetics）一天HUVEC和一天前珠串成纤维细胞前嵌入。 </li><li> 〜400人脐静脉内皮细胞，每珠浓度是必要的。 </li><li> 20,000元以及成纤维细胞是必要的。 </li></ol><p class="jove_step">涂层珠与人脐静脉内皮细胞 - 天-1 </p><ol><li> Trypsinize人脐静脉内皮细胞。 </li><li>允许珠解决（请勿离心机！?...

<ol>
	<li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microcarrier-based+cocultivation+system+for+the+investigation+of+factors+and+cells+involved+in+angiogenesis+in+three-dimensional+fibrin+matrices+in+vitro.">A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro.</a> <em style='font-style: italic'>Histochem Cell Biol</em>. <b>104</b>, 459-466 (1995).</li><li>Nehls, V., Drenckhahn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+microcarrier-based+in+vitro+assay+for+rapid+and+reliable+quantification+of+three-dimensional+cell+migration+and+angiogenesis.">A novel, microcarrier-based in vitro assay for rapid and reliable quantification of three-dimensional cell migration and angiogenesis.</a> <em style='font-style: italic'>Microvasc Res</em>. <b>50</b>, 311-322 (1995).</li></ol>

<p>有越来越多的共识，三维（3D）在体外血管生成的实验提供了一个模型，它在体内更接近实际环境比可达到使用2D文化。很明显，优越的3D系统应重现性好，能够模仿一些血管生成的主要步骤。虽然前几届的3D检测得到了发展，其中许多可以使用难以取得微血管细胞，或仅复述一些阶段。在这个视频中，我们描述和执行在体外血管生成试验，利用人脐静脉EC，这是很容易获得和在血管研究中最常用的EC的优化。该试?...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Cytodex-3 Beads</td>
<td>Reagent</td>
<td>Amersham Pharmacia</td>
<td>17-0485-01</td>
<td>10 g/bottle. 
1.	0.5 g of dry beads are hydrated and swollen in 50 mL PBS (pH=7.4) for at least 3 hours at RT.
Use a 50 mL tube and place it on the rocker.
2.	Let the beads settle down (~ 15 min). Discard the supernatant and wash the beads for a few minutes in fresh PBS (50 mL).
3.	Discard the PBS and replace with fresh PBS:
25 mL -> 20 mg/mL => 60000 beads/mL or
50 mL -> 10 mg/mL => 30000 beads/ mL
4.	Place the bead suspension in a siliconized glass bottle (Windshield Wiper or Sigmacote).
5.	Sterilize the beads by autoclaving for 15 min at 115&#xF0B0;C.
6.	Store it at 4&#xF0B0;C.
</td>
</tr>
<tr>
<td>Aprotinin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-1153</td>
<td>10mg/bottle. Reconstitute lyophilized aprotinin at 4 U/mL in DI water. Sterile filter. Make aliquots of 1 mL each. Store at -20C.
</td>
</tr>
<tr>
<td>Fibrinogen Type I</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8630</td>
<td>1g. Dissolve 2 mg/mL fibrinogen in DPBS
Note clottable protein % and adjust accordingly 
Heat in a 37&#xF0B0;C-water bath to dissolve the fibrinogen. Mix by inverting the tube. Do not vortex.
Sterile filter through 0.22 um
</td>
</tr>
<tr>
<td>Thrombin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T-3399</td>
<td>22 mg=1000 units. Reconstitute in sterile water at 50 U/mL. Make aliquots of 0.5 mL each. Store at -20C.
</td>
</tr>		</tbody>
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optimized fibrin gel bead assay for the study of angiogenesis

<p class="jove_content">血管生成是一个复杂的多步骤的过程，在血管生成刺激，新船都从现有的血管创建。这些步骤包括：降解基底膜，增殖和迁移（发芽）内皮细胞到细胞外基质，成线对齐欧共体（EC），分支，管腔形成，吻合术，并形成一个新的基底膜。在体外实验中有许多已开发研究这个过程，但大多只模仿血管生成的某些阶段，形态内检测的船只往往并不像在体内的船只。早期Nehls和Drenckhahn工作的基础上，我们进行了优化，利用人脐静脉EC和成纤维细胞在体外血管生成试验。这种模式概括所有的血管生成的关键早期阶段，重要的是，船只显示极化欧共体包围专利间流明。欧共体涂到cytodex微载体，并嵌入到纤维蛋白凝胶。成纤维细胞分层他们提供必要的可溶性因子，促进欧共体从珠的表面发芽的凝胶上。几天后，无数的船只，可以很容易地相衬和时间推移显微镜下观察。本视频演示了在建立这些文化的关键步骤。</p>

Watch this Scientific Journal Video about Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis at JoVE.com

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis (Video) | JoVE

<p class="jove_content">本视频演示了一个协议，在体外血管生成试验，概括的血管生成的几个阶段。显示图像时间的推移，发芽，管腔形成，分支和吻合 - 血管生成的关键特征 - 。</p>

优化的纤维蛋白凝胶珠检测血管生成的研究

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