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。

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

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

用于血管生成研究 的改良体内 基质凝胶塞测定

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as embryonic development or curing atherosclerosis, tumor development, etc. The construction of a reliable model to evaluate the extent of angiogenesis is critical for myocardial research or the discovery of medicines for angiogenesis-depend diseases.However, most of the existing models are complex, expensive, and require specialized equipment. Here, we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining in a simple, more intuitive, as well as efficient manner.

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

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。

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

549 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

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

科研

生物学

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Begin by preparing the complete endothelial culture medium using 460 milliliters of endothelial cell medium, and adding into it, 50 milliliters of FBS, five milliliters of penicillin streptomycin, and five milliliters of endothelial cell growth supplement. The prepared media can be stored at four degrees celsius for a month. Next, in a 100 millimeter tissue culture dish containing 0.1 million vascular cells at eight milliliters of the prepared complete endothelial culture medium and culture the cells at 37 degrees Celsius and 5%carbon dioxide until 70%co-fluency is achieved.Then, remove the culture medium from the dish and rinse the cells twice with PBS to eliminate unattached cells and debris. After removing the PBS, add three milliliters of 0.25%trypsin, containing 2.21 millimolar EDTA to the cells and incubate the dish at 37 degrees Celsius for one minute. Verify cell detachment under a light microscope at 40 times magnification.Neutralize the trypsin with seven milliliters of complete endothelial culture medium and gently rinse the cells off the culture dish. Centrifuge the cell suspension after collecting it in a 15 milliliter tube at 400G for 10 minutes. Re-suspend the cells in five milliliters of complete endothelial culture medium after removing the supernatant, then count the cells using a hemo-cytometer, and transfer a calculated volume of suspension containing 2 million cells to a sterile 1.5 milliliter tube.Pellet the cells by centrifuging the suspension at 400G for five minutes before removing the supernatant. The vascular cells are now ready for mixing with the matrix gel for matrix gel plug assay.

制备用于体内基质凝胶塞测定的血管细胞

Preparing Matrix Gel with Vascular Cells for In Vivo Matrix Gel Plug Assay

To begin, prepare 10 times M199 culture medium by dissolving M199 powder to 10 times concentration in 90 milliliters of deionized water, followed by the addition of 10 milliliters of FBS. Filter the solution through a 0.22 micron filter. The filtered medium can be stored at four degrees Celsius for up to two months.For preparing the matrix gel with vascular cells, use about 2 million vascular cells. Cool the sterile 1.5 milliliter tube containing the appropriately prepared cell pellet in an ice bath. Thaw the matrix gel completely in an ice bath without mixing or vortexing it.Similarly, cool the prepared 10 times M199 culture media along with the test reagent or drug of interest to four degrees Celsius. Next, maintaining a volume ratio of 8.8 to 1 to 0.2, combine the pre-cooled matrix gel with the pre-cooled 10 times M199 media containing 10%FBS, and the pre-cooled test reagent to produce the matrix gel mixture. Finally, re-suspend the pre-cooled cells in 400 microliters of the prepared matrix gel mixture by mixing gently to avoid forming bubbles.Keep the tube containing the cells in matrix gel on ice until the mice are ready for the matrix gel injection.

制备带有血管细胞的基质凝胶用于体内基质凝胶塞测定

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

To begin, place the 30 gauge one milliliter insulin syringes in a four degrees Celsius refrigerator for cooling. Keep the freshly prepared matrix gel mix containing vascular cells on ice until the mouse is ready for the injection. Next, place the properly anesthetized mouse on the operating board in a prone position and secure its limbs onto the board using tape.Load a pre cooled insulin syringe with 300 microliters of the matrix gel mixture, avoiding the formation of bubbles. Disinfect the skin on the back of the mouse using 75%alcohol pads, then subcutaneously inject 300 microliters of the matrix gel mixture in the syringe into one side of the animal's back. Gently remove the needle from the injection site to prevent any leakage of the gel mixture.Ensure the formation of a small hump at the injection site. Position the mouse on a heating pad for two minutes to allow the matrix gel mixture to coagulate and form a plug. Repeat the demonstrated steps to create a plug on the other side of the mouse's back.Mark the edges of the hump using a marker pen. House the animal in a specific pathogen free experimental animal laboratory at 20 to 24 degrees with 12 hour light and 12 hour dark cycles for seven days.

将基质凝胶混合物注射到小鼠中进行基质凝胶塞测定

Dextran-FITC Injection and Matrix Gel Plug Collection from Mouse for Matrix Gel Plug Assay

To inject the mouse with Dextran, FITC seven days post the matrix gel injection, RESUSPEND 0.5 milligrams of Dextran, FITC in 500 microliters of double distilled water. Thoroughly vortex it to obtain a one microgram per microliter Dextran, FITC solution. Fill a 29 gauge syringe with 50 microliters of the Dextran FITC solution.Secure the mouse on the tail vein injection instrument, sanitize the tail with a 75%alcohol soaked cotton ball before carefully injecting 50 microliters of the Dextran FITC solution in the syringe into the tail vein. Apply pressure to the injection site with the cotton swab for one minute to staunch bleeding. Return the mouse to its cage and let it relax for 30 minutes before collecting the matrix gel plug.To collect the gel plug, cut the skin of the euthanized mouse along the marked border of the plug, using surgical scissors. And remove the skin above the matrix gel plug. Collect the plug and rinse it in a beaker with phosphate buffered saline to wash off any excess blood.Next, cover the bottom of the tissue embedding cassette with about 0.5 milliliters of tissue embedding gel. Then swiftly place the matrix gel plug on the tissue embedding gel in the planned orientation. Embed the plug with additional tissue embedding gel.Solidify the cassettes at minus 80 degrees Celsius for 12 hours. After removing the solidified plug block, cut 12 micron thick sections using a freezing microtome. Then mount the sections onto microscope slides.