| 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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| Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons
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Author:
Date:
2016-04-05
[Abstract] We here provide a detailed protocol for the isolation and culture of primary mouse sensory neurons. The cell bodies of sensory afferent pseudounipolar neurons are located in dorsal root ganglia (DRGs) along the vertebral column. Dissected mouse DRGs can be dissociated into single cells by enzymatic digestion to obtain primary cultures of mouse sensory neurons as performed in the studies reported by Khaminets et al. (2015).
[摘要] 我们在这里提供了详细的协议，用于隔离和培养的主要小鼠感觉神经元。 感觉传入假性极化神经元的细胞体位于沿着脊柱的背根神经节（DRG）中。 解离的小鼠DRG可以通过酶消化解离成单个细胞，以获得小鼠感觉神经元的原代培养物，如Khaminets等人（2015）报道的研究中所进行的。
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| FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
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Author:
Date:
2015-08-20
[Abstract] Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal ...
[摘要] 荧光活化细胞分选（FACS）是用于从成年小鼠骨骼肌纯化卫星细胞或肌肉干细胞的灵敏和精确的方法（Liu等人，2013; Sacco等人， ，2008; Tierney ，，2014）。 后肢肌肉的机械和酶消化将单核肌细胞释放到悬浮液中。 该方案采用分级分离策略，通过磁性分离和基于FACS的排除，耗尽表达细胞表面标记CD45，CD31，CD11b和Ly-6A/E-Sca1的细胞，并阳性选择表达α7-整联蛋白和CD34的细胞。 这使得研究者能够成功地富集均匀表达配对盒转录因子Pax7并且能够长期自我更新，骨骼肌修复和肌肉干细胞池再增殖的卫星细胞。
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