这项工作是由NCI R01 CA120659（RS）的支持。

1。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<ol><li>所有的肿瘤细胞和微血管内皮细胞的制备：U87细胞，黑色素瘤细胞，乳腺癌MDA - MB - 435，和结肠癌细胞HCT116 B16F1如脑肿瘤细胞生长，补充10％胎牛血清和青霉素/链霉素的DMEM（Invitrogen公司）。内皮细胞株人微血管（HMVECs）内皮细胞培养EBM2媒介为辅1微克/毫升氢化可的松和1 ng / ml的表皮生长因子，10％胎牛血清和青霉素/链霉素（龙沙）。细胞用PBS洗3次，并用0.05％胰酶/ EDTA（Invitrogen）的分离。在离心5分钟转速2,000后，细胞沉淀用PBS冲洗一遍。然后，颗粒细胞与一个血球计数。 </li><li>基质胶准备：一个等分的增长因素减少基质胶（BD生物科学）温暖在室温。之前完全解冻，它被转移到冰和液体放在冰上至少10分钟。镀96孔板50μL基质胶基质胶均匀分布在一个水平位置，让，并在37 ° C孵育30 min </li><li>管形成细胞（1-2 × 10 4）重新悬浮无血清DMEM肿瘤细胞或血管内皮细胞的EBM - 2，并装上基质胶的顶部。如果这个实验是用来测量肾小管发展一些代理商的影响，这些代理商（刺激或抑制），分别加入无血清培养基。每个条件组中包含4-6井。 </li><li>图像和数据分析：在37℃以下孵化° C过夜，直接在显微镜下观察分析每口井。如果需要固定管，轻轻加入100μL10％的福尔马林生理盐水为基础的解决方案和十个分钟后，有人分析准备。 10X相衬显微镜下，在每个字段管成像和计算平均每口井从3-5随机领域肾小管。 </li></ol> 2。代表性的成果： HMVECs被用来作为阳性对照，这些管明确拉长胞体连接，形成多边形网络开发。 B16F1，U87，和MDA - MB - 435细胞发育类似HMVECs形成的血管管，但没有HCT116细胞（图1）。一个细胞剂量依赖性管形成U87细胞中进行了测试。图2表明，万细胞形成停产肾小管。一旦细胞一倍，是一个坚实的血管网与HMVECs看到。与此相反，细胞小于5,000未能形成血管的表型。 <img src="/files/ftp_upload/3040/3040fig1.jpg" alt="图1"/> 图1 管形成HMVECs，U87，MDA - MB - 435，和B16F1细胞，但诱导HCT116细胞的所有细胞（2 × 10 4）被装上基质胶和孵育过夜。管使用相衬成像。 HMVECs被用来作为阳性对照。显示3-5领域的代表。酒吧：100微米。 <img src="/files/ftp_upload/3040/3040fig2.jpg" alt="图2"/> 用于管形成图2。U87细胞诱导的小管在细胞数量依赖性。U87细胞，在弯道中表示不同的电话号码。 HMVECs用于阳性对照。酒吧：100毫米。

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没有利益冲突的声明。

为了这个实验取得成功，基质胶的质量，应先进行测试。一个小样本，可从屋宇署生物科学，预先使用HMVECs运行检测。不同批次的产品可能会显示不同的素质，其中一些地段不提供管形成的最佳条件。第二，任何气泡应避免在96孔板中装入基质胶等份，因为泡沫可以破坏肾小管形成。如果发现在一个良好的微小气泡，基质胶可以立即移动返回到原来的基质胶管在实验过程中应保持在冰上。此外，?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> DMEM培养液</td><td> Invitrogen公司</td><td> 11995 </td></tr><tr><td>胎牛血清</td><td> Invitrogen公司</td><td> 16000-044 </td></tr><tr><td>生长因子减少基质胶</td><td> BD生物科学</td><td> 47743-720 </td></tr><tr><td> EBM2套件</td><td>龙沙</td><td> CC - 3156 </td></tr><tr><td>尼康ECLIPSE TS100显微镜</td><td>尼康</td><td></td></tr></tbody></table>

a matrigel-based tube formation assay to assess the vasculogenic activity of tumor cells

在过去的几十年里，一个管形成实验使用的增长因素，减少基质胶通常已经证明在 体外培养1-5的血管内皮细胞的血管生成活性。然而，最近越来越多的证据表明，这种检测不仅限于测试对血管内皮细胞的行为。相反，它也被用来测试的肿瘤细胞数量的能力，培养血管表型 6-8 。这种能力是与他 ​​们的血管生成xenotransplanted动物，一个过程被称为血管生成拟态（VM）的9所确定的行为是一致的。有大量的证据表明，肿瘤细胞介导的虚拟机中起着至关重要的作用，在肿瘤的发展，独立于血管内皮细胞血管生成6， 10-13。例如，肿瘤细胞被发现参与组织样本中的血液灌流，血管通道形成黑色素瘤和胶质母细胞瘤患者，8，10，11 。在这里，我们描述了这种管状网络检测，作为一个有用的工具，在评价肿瘤细胞的血管生成活性。我们发现，一些肿瘤细胞株，如黑色素瘤B16F1细胞，胶质母细胞瘤U87细胞和乳腺癌MDA - MB - 435细胞能够形成血管管;但也有一些如结肠癌HCT116细胞。此外，这种血管表型是依赖于基质胶上镀的手机号码。因此，这种检测可以作为强大的工具，屏幕上的多种细胞类型，包括血管细胞，肿瘤细胞以及其它细胞的血管潜力。

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

管形成实验是用来评估肿瘤细胞的血管活性。

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 ...

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65.8K 次观看.  University of Massachusetts. 管形成实验是用来评估肿瘤细胞的血管活性。