| Isolation of Microvascular Endothelial Cells
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Author:
Date:
2018-06-20
[Abstract] The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. Murine endothelial cell culture is an important tool for cardiovascular disease research. This protocol demonstrates a quick, efficient method for the isolation of microvascular endothelial cells from murine tissues without any special equipment. To isolate endothelial cells, the lung or heart were mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained was then incubated with an anti-CD31, anti-CD105 antibody and with biotinylated isolectin B-4. The endothelial cells were harvested using magnetic bead separation with rat anti-mouse Ig- and streptavidin-conjugated microbeads. Endothelial cells were ...
[摘要] 血管内皮是正常血管稳态所必需的。其功能障碍参与各种心血管疾病。小鼠内皮细胞培养是心血管疾病研究的重要工具。该协议演示了一种快速，有效的方法从小鼠组织中分离微血管内皮细胞而无需任何特殊设备。为了分离内皮细胞，将肺或心脏机械切碎并用胶原酶和胰蛋白酶进行酶促消化。然后将获得的单细胞悬浮液与抗CD31，抗CD105抗体和生物素化的异凝集素B-4温育。使用大鼠抗小鼠Ig-和链霉亲和素缀合的微珠，使用磁珠分离收获内皮细胞。扩张内皮细胞并收集用于随后的分析。这些培养物的形态和表型特征在培养10代以上保持稳定。在任何阶段没有过度生长的非内皮来源的污染细胞。
【背景】微血管内皮细胞通过调节白细胞再循环和作为T淋巴细胞的抗原呈递细胞而在免疫应答的发展中起中心作用。内皮的良好状态对血管稳态很重要。功能失调的内皮参与各种心血管疾病，包括动脉粥样硬化，血管炎和缺血/再灌注损伤（Cid et al。，2004; Wang等人，2007）。因此，体外培养的内皮细胞培养物是研究血管生理学和疾病病理学的重要工具。然而，分离原代鼠类内皮细胞被认为是特别困难的，因为大多数描述的方案涉及用消化酶灌注器官或大血管和耗时的纯化过程（Gumkowski等人，1987） 。
该协议的目的是提供一种简单的方法，不需要使用任何特殊设备即可从肺/心脏中分离和扩展内皮细胞。使用这种方法，我们以前补充了体内研究，证明了CD31信号传导在内皮细胞细胞保护中的重要性（Cheung等人，2015年）。 ...
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| In vivo Extravasation Assay
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Author:
Date:
2014-02-20
[Abstract] Tumor metastases develop when disseminated intravascular cancer cells acquire the ability to arrest by adhering to the capillary walls of distant organs, actively extravasate into their parenchyma, proliferate and establish secondary colonies. The extravasation assay described here is an in vivo technique aimed to analyze the ability of tumor cells to achieve early colonization of the lungs following tail vein injection in mice. Importantly, tumor cells need to be easily visible, therefore either they are fluorescent (e.g. expressing RFP or GFP) or they have to be pre-labelled with a fluorescent tracker prior to injection. Lungs are analyzed at different time points, experimentally determined by the researcher, depending on cell features and malignancy. Generally, an ...
[摘要] 当弥散性血管内癌细胞通过粘附到远端器官的毛细血管壁，主动外渗到其实质中，增殖和建立次级集落而获得阻滞的能力时，肿瘤转移发展。这里描述的外渗测定是体内技术，其目的在于分析肿瘤细胞在小鼠尾静脉注射后实现早期肺定植的能力。重要的是，肿瘤细胞需要容易可见，因此它们是荧光的（例如表达RFP或GFP），或者它们必须在注射之前用荧光跟踪器预标记。根据细胞特征和恶性肿瘤，在不同的时间点分析肺，由研究者实验确定。通常，需要早期时间点来检查针对注射的各种细胞的肺血管系统中的相等倒伏。在一个或多个稍后的时间点（6至48小时），定量分散在肺实质中的外渗细胞。使用我们的方案外渗在整个肺中直接评估离体考虑细胞荧光。然而，推荐内皮标记物的免疫荧光染色和肺切片的显微镜分析以评价肿瘤细胞（即在血管内或与其相关联的单个细胞或簇）内部，外部的定位和状态。由于外渗不仅受肿瘤细胞运动性而且受其存活能力的影响，用这种技术获得的结果应当与增殖和凋亡分析互补。
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| Isolation of Endothelial Cells from Mice
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Author:
Date:
2012-04-20
[Abstract] Endothelial cells line the entire circulatory system, from the heart to the smallest capillary. In this protocol, attaining and maintaining vitality of the organ is critical. Minimizing the processing time between removing the organ and beginning the digestions will improve yields as cells are more viable and are not under hypoxic conditions for long. For this protocol to be most effective, mice should be 5-6 weeks of age. Older mice result in lower yields of endothelial cells. In our hands, as few as 5 mice can result in good yields.
[摘要] 内皮细胞分布整个循环系统，从心脏到最小的毛细血管。 在这个协议中，获得和保持器官的活力是至关重要的。 最小化去除器官和开始消化之间的处理时间将提高产量，因为细胞更可行并且长时间不处于低氧条件下。 为了使该方案最有效，小鼠应当是5-6周龄。 老年小鼠导致内皮细胞的产量降低。 在我们手中，少至5只小鼠可以产生良好的产量。
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