这项工作由浙江省自然科学基金（LY22H020005）和中国国家自然科学基金（81873466）资助。

所有涉及动物受试者的程序均已获得温州医科大学机构动物护理和使用委员会（IACUC）的批准（XMSQ2021-0057,2021 年 7 月 19日）。所有试剂和耗材都列在 材料表中。
1.培养基制备
<ol><li>10x M199 培养基：将 M199 粉末用 90 mL 去离子水溶解至 10 倍浓度，加入 10 mL 胎牛血清 （FBS），然后通过 0.22 μm 过滤器。将培养基储存在4°C长达2个月。</li>
	<li>完全内皮培养基：向 460 mL 内皮细胞培养基 （ECM） 中加入 50 mL FBS、5 mL 青霉素/链霉素和 5 mL 内皮细胞生长补充剂。将培养基储存在4°C长达1个月。</li>
</ol>2. 血管细胞制备
<ol><li>在37°C和5%CO2至70%汇合度的100mm组织培养皿中，用8mL完全内皮培养基培养1×105个血管细胞（原代培养的内皮祖细胞14，内皮细胞或内皮细胞系）。</li>
	<li>取出培养基，用 1x 磷酸盐缓冲盐水 （PBS） 冲洗培养皿 2 次，以去除未附着的细胞和碎屑。去除PBS并加入3mL含有2.21mM EDTA的0.25%胰蛋白酶，并在37°C孵育1分钟。</li>
	<li>用 7 mL 完全内皮培养基中和胰蛋白酶，然后轻轻冲洗培养皿中的细胞。在光学显微镜（40倍放大倍率）下确认细胞分离。</li>
	<li>将细胞悬液收集在 15 mL 管中，并以 400 x g 离心 10 分钟。除去上清液并用 5 mL 完全内皮培养基重悬细胞。</li>
	<li>使用血细胞计数器计数细胞，并将含有 2 x 106 个细胞的悬浮液移至无菌 1.5 mL 管中。每个插头包含 1.5 x 106 个电池，另外 25% 的电池用于浪费。每组至少包括 3 个插头。 注意：经过试验，发现 300 μL 基质凝胶中的 1.5 x 106 个细胞导致血管生成的正常发育，因此选择进行实验。</li>
	<li>将细胞悬浮液以400× g 离心5分钟以沉淀细胞，然后除去上清液。</li>
</ol>3.基质凝胶制备
<ol><li>在4°C水浴中预冷含有细胞沉淀的无菌1.5mL管。在4°C冰箱中预冷30G 1mL胰岛素注射器。</li>
	<li>在4°C水浴中完全解冻基质凝胶。不要混合或涡旋。在4°C水浴中预冷10x M199和测试试剂/目标药物。</li>
	<li>将预冷的基质凝胶与含有10%FBS的10x M199和待测试剂混合，体积比为8.8：1：0.2，得到含有1%FBS的基质凝胶和M199混合物和试剂。</li>
	<li>用 400 μL 基质凝胶混合物重悬细胞，轻轻混合以避免形成气泡。将试管放在冰上，直到小鼠准备注射。将基质凝胶混合物始终放在冰上以避免凝固。 注意：在这里，使用含有 1.5 x 106 个细胞的 300 μL 基质凝胶混合物形成凝胶塞。根据步骤2中25%的额外细胞制备25%的额外凝胶。</li>
</ol>4.小鼠准备
<ol><li>用异氟醚（3%异氟醚在100%氧气中以1 L / min的流速）麻醉6-8周龄的雄性Nu / Nu小鼠（18-25g）。麻醉成功后，通过没有矫正反射和脚趾捏反射确认，将小鼠移出腔室，将小鼠移出腔室并在小鼠上放置麻醉面罩，并将异氟烷的浓度更改为1.5%在100%氧气中以1L / min的流速。 注意：在整个过程中使用加热垫为动物提供热支持。</li>
	<li>在眼睛上使用兽医软膏以防止干燥。将小鼠的四肢以俯卧位粘在操作板上。</li>
</ol>5.基质凝胶混合物注射
<ol><li>用 30G 针头将 300 μL 基质凝胶混合物加载到 1 mL 胰岛素注射器中。避免气泡形成。快速加载基质凝胶（2分钟内），以避免在此期间凝固。立即将装有基质凝胶的胰岛素注射器放在冰上。</li>
	<li>使用75%的酒精棉片清洁小鼠背部的皮肤。皮下注射300μL基质凝胶混合物到小鼠背部的一侧。</li>
	<li>轻轻地从注射部位取下针头，以防止基质凝胶混合物泄漏。检查注射部位是否有小驼峰（图1）。</li>
	<li>将鼠标放在加热垫上2分钟，让基质凝胶混合物凝结并形成塞子。</li>
	<li>重复步骤 5.2。至 5.4.在鼠标背面的另一侧创建插头。用记号笔标记驼峰的边缘。</li>
	<li>观察小鼠，直到它恢复足够的意识以保持胸骨卧位。将鼠标放在单独的笼子中，直到它完全恢复。将小鼠饲养在22±2°C的SPF级实验动物实验室中，光/暗循环12小时，持续7天。</li>
</ol>6.通过尾静脉注射葡聚糖-FITC
<ol><li>基质凝胶注射7天后，将0.5mg葡聚糖-FITC与500μL双蒸水（ddH2O）重悬，然后剧烈涡旋，得到终浓度为1μg/μL的葡聚糖-FITC溶液。 注意：使用分子量为~150 kDa的葡聚糖-FITC进行血管生成测定，并使用分子量为~4 kDa的葡聚糖-FITC进行血管通透性测定。</li>
	<li>将 50 μL 葡聚糖-FITC 溶液加载到 29G 注射器中。</li>
	<li>将鼠标固定在尾静脉注射仪器上。用75%酒精棉球清洁尾部，并通过尾静脉轻轻注射50μL葡聚糖-FITC。</li>
	<li>用棉签按压注射部位1分钟以止血，然后将小鼠放回笼子中30分钟。</li>
	<li>重复步骤 6.3。和 6.4.直到所有小鼠都接受葡聚糖-FITC注射。</li>
</ol>7. 基质凝胶塞收集
<ol><li>腹膜内注射 1% 戊巴比妥钠在 PBS（200 mg/kg 体重）中，并使用 IACUC 批准的程序对小鼠实施安乐死。</li>
	<li>使用手术剪刀沿栓子的标记边缘切割皮肤，然后去除基质凝胶塞上方的皮肤。</li>
	<li>收集塞子并在装有1x PBS的小烧杯中冲洗以洗去多余的血液（图2A）。栓子的颜色可以大致描绘出血液丰度和新血管的内容（图3A）。</li>
</ol>8. 嵌入基质凝胶塞和切片制备
<ol><li>用约0.5 mL的组织包埋凝胶覆盖组织包埋盒的按钮，然后立即将基质凝胶塞以所需方向放置在组织包埋凝胶上，如 图2B所示。然后，用额外的组织包埋凝胶包埋塞子。</li>
	<li>将盒放入-80°C冰箱中12小时凝固。</li>
	<li>对于厚切片制备，取出凝固的塞块，按照说明使用冷冻切片机切割12μm厚的切片，然后安装在显微镜载玻片上。</li>
	<li>从每个插头块上切下 5-10 个部分。</li>
</ol>9.血管生成的量化（图3）
<ol><li>在10倍放大倍率下，使用488nm波长的倒置荧光显微镜从切片的5个独立场获取荧光图像。</li>
	<li>使用 Image J 软件 （https://imagej.en.softonic.com/） 打开图像文件，然后单击 “血管生成分析 ”按钮。然后，单击 “分析 HUVEC 相差 ”按钮，并切换到 “统计结果表” 以收集信息。测量每个切片的所有 5 张采集的荧光图像。</li>
	<li>分析不同组新血管的总长度，以统计评估这些组之间血管生成的差异。 注：总主段长度表示新船的总长度。增加新血管长度的试剂称为促血管生成剂，而减少新血管长度的试剂称为抗血管生成。</li>
</ol>10.血管通透性的量化（图4）
<ol><li>在20倍放大倍率下，使用488nm波长的倒置荧光显微镜从切片的5个独立场获取荧光图像。</li>
	<li>使用 Image J 软件打开图像文件。单击 “手绘选择 ”按钮并圈出泄漏区域，然后单击 “分析 ”菜单并选择“ 测量 ”选项以获取泄漏区域的区域信息。使用渗漏面积和图像面积的比率来指示血管通透性。</li>
	<li>分析不同组的渗漏面积和图像面积的比率，以统计评估这些组之间血管通透性的差异。</li>
</ol>

使用改良基质凝胶塞测定简化血管生成研究

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作者声明没有利益冲突。

我们提出了一种可靠且方便的方法，用于在不染色 的情况下定量评估体内 血管生成。在该方案中，在促血管生成或抗血管生成试剂存在下将血管细胞与基质凝胶混合，然后皮下注射到Nu / Nu小鼠的背部以形成凝胶塞（图1）。凝胶栓形成7天后，静脉注射葡聚糖-FITC循环30分钟。收集凝胶塞并用组织包埋凝胶包埋，并切片 12 μm 用于摄影（图 2）。葡...

血管生成是从预先存在的血管形成新血管的过程，在许多生理和病理过程中起着至关重要的作用，例如胚胎发育、伤口愈合、动脉粥样硬化、肿瘤发展等1,2,3,4,5。这种动态过程涉及几个步骤，包括基质的降解、血管细胞增殖、迁移和自组织以形成管状结构以及新血管的稳定 6。促进血管生成已被证明在治疗心肌梗塞、中风和其他类型的缺血性疾病中至关重要7，而抑制血管生成被认为是治疗癌症8和类风湿性疾病的有前途的策略9。血管生成被认为是药物发现的组织原则10.因此，构建一种可靠且方便的方法来评估血管生成的程度对于血管生成依赖性疾病的机械研究或药物发现至关重要。
已经开发了几种体外和体内模型来评估血管生成11。其中，二维（2-D）模型，如基质凝胶管形成测定12，不能形成功能性管状结构。动物模型，如后肢缺血模型13,14，可以再现血管生成过程，但很复杂，需要激光散斑血流成像系统。血管形态发生的 3D 模型，如基质凝胶塞测定，提供了一个简单的平台，可以模拟体内血管生成的过程 15，但血管生成的检测需要免疫组化或免疫荧光染色 16,17,18，这些方法可变且可视化不佳。
在这里，我们描述了一种改良的基质凝胶塞测定方案，其中将血管细胞与基质凝胶混合并皮下注射到小鼠背部以形成塞子。在塞子中，血管细胞需要降解基质、增殖、迁移和自组织，最终形成内部环境中有血流的功能性血管。此后，通过尾静脉注射荧光标记的葡聚糖，流经塞子，并将标记可视化以指示新血管。血管生成的内容可以通过血管的长度来定量评估。该方法可以形成在二维血管生成模型12中无法产生的功能性血管，并且不需要像普通基质凝胶塞测定11那样复杂的染色过程。它也不需要昂贵的特定仪器，如后肢缺血模型13、14、19 中的激光散斑血流成像系统。该方法用途广泛、成本低、可量化且易于执行，可用于确定药物的促血管生成或抗血管生成能力，或用于涉及血管生成的机械研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adhesion Microscope Slides</td>
			<td>CITOTEST</td>
			<td>188105</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia System</td>
			<td>RWD</td>
			<td>R640-S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoslicer</td>
			<td>Thermo Fisher</td>
			<td>CryoStar NX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans-FITC-150kDa</td>
			<td>WEIHUA BIO</td>
			<td>WH007N07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrans-FITC-4kDa</td>
			<td>WEIHUA BIO</td>
			<td>WH007N0705</td>
			<td></td>
		</tr>
		<tr>
			<td>Embedding Cassettes</td>
			<td>CITOTEST</td>
			<td>80203-0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium</td>
			<td>ScienCell</td>
			<td>35809</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Growth Supplements</td>
			<td>ScienCell</td>
			<td>1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10100147C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Factor 1</td>
			<td>AtaGenix</td>
			<td>9043p-082318-A01</td>
			<td>FGF1</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ni</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Boruida</td>
			<td>30-50-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin Syringe</td>
			<td>BD</td>
			<td>300841</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory&#160;Balance</td>
			<td>Sartorius</td>
			<td>BSA124S-CW</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Matrix gel</td>
		</tr>
		<tr>
			<td>Medium 199 powder</td>
			<td>Gibco</td>
			<td>31100-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature (OCT) Compound</td>
			<td>SUKURA</td>
			<td>4583</td>
			<td>Tissue embedding gel</td>
		</tr>
		<tr>
			<td>Palmitate Acid</td>
			<td>KunChuang</td>
			<td>KC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Liquid</td>
			<td>Solarbio</td>
			<td>P1400</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Solarbio</td>
			<td>P1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>RWD</td>
			<td>RWD</td>
			<td></td>
		</tr>
		<tr>
			<td>Tail Vein Injection Instrument</td>
			<td>KEW BASIS</td>
			<td>KW-XXY</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution</td>
			<td>Solarbio</td>
			<td>T1320</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Temperature Freezer</td>
			<td>eppendorf</td>
			<td>U410</td>
			<td></td>
		</tr>
		<tr>
			<td>Vascular Endothelial Growth Factor</td>
			<td>CHAMOT</td>
			<td>CM058-5HP</td>
			<td>VEGF</td>
		</tr>
	</tbody>
</table>

modified in vivo matrix gel plug assay for angiogenesis studies

已经开发了几种模型来研究 体内血管生成。然而，这些模型大多复杂且昂贵，需要专门的设备，或者难以进行后续定量分析。在这里，我们提出了一种改良的基质凝胶塞测定法来评估 体内血管生成。在该方案中，在存在或不存在促血管生成或抗血管生成试剂的情况下，将血管细胞与基质凝胶混合，然后皮下注射到受体小鼠的背部。7天后，通过尾静脉注射含有葡聚糖-FITC的磷酸盐缓冲盐水并在血管中循环30分钟。收集基质凝胶塞并用组织包埋凝胶包埋，然后切割 12 μm 切片用于荧光检测，无需染色。在该测定中，高分子量（~150,000 Da）的葡聚糖-FITC可用于指示功能性血管以检测其长度，而低分子量（~4,400 Da）的葡聚糖-FITC可用于指示新血管的渗透性。总之，该方案可为 体内血管生成的定量研究提供一种可靠、方便的方法。

图1 是描述如何制备基质凝胶、血管细胞、培养基和试剂混合物的流程图。然后将混合物皮下注射到Nu / Nu小鼠的背部，并使用加热垫加热以加速其凝固，最终形成凝胶塞。
图2A 是指示荧光标记葡聚糖的血管的流程图。通过尾静脉和圆圈注射荧光标记的葡聚糖30分钟，使其可以进入凝胶塞中的功能血管。此后，收集凝胶栓，使用组织...

Watch this Scientific Journal Video about Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies at JoVE.com

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

这里介绍的方法可以在不染色的情况下评估 试剂对体内 血管生成或血管通透性的影响。该方法使用右旋糖酐-FITC通过尾静脉注射来观察新血管或血管渗漏。

用于血管生成研究 的改良体内 基质凝胶塞测定

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

我们的研究涉及血管生成，即从现有血管形成新血管的过程。它在许多生理和病理过程中起着关键作用，如胚胎发育或治疗动脉粥样硬化、肿瘤发展等。构建一个可靠的模型来评估血管生成的程度对于心肌研究或发现治疗血管生成依赖性疾病的药物至关重要。然而，大多数现有模型都复杂、昂贵，并且需要专门的设备。在这里，我们提出了一种改良的基质凝胶塞测定法，可用于以简单、更直观和有效的方式研究血管生成和血管通透性，而无需染色。

modified-in-vivo-matrix-gel-plug-assay-for-angiogenesis-studies

Research

JoVE Journal

Biology

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

788 次观看.  Pingyang Affiliated Hospital of Wenzhou Medical University. 我们的研究涉及血管生成，即从现有血管形成新血管的过程。它在许多生理和病理过程中起着关键作用，如胚胎发育或治疗动脉粥样硬化、肿瘤发展等。构建一个可靠的模型来评估血管生成的程度对于心肌研究或发现治疗血管生成依赖性疾病的药物至关重要。然而，大多数现有模型都复杂、昂贵，并且需要专门的设备。在这里，我们提出了一种改良的基质凝胶塞测定法?...

视频: 作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

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 ...

科研

生物学

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

大鼠肠系膜血管生成法

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

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

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

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

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

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 ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

染色质免疫沉淀拟南芥蛋白-DNA相互作用的鉴定<em&gt;在体内</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

体内碱性彗星试验和酶修饰碱性彗星试验测量DNA链断裂和DNA氧化损伤大鼠肝

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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.

在人类内皮周细胞的相互作用使用基质凝胶插件检测小鼠的体内研究

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

体外细胞生理学研究中视网膜微动脉的分离

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

改进的高通量DNA提取板加载方法，降低污染的可能性

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...

用于血管生成研究 的改良体内 基质凝胶塞测定

用于血管生成研究的改良体内基质凝胶塞测定