| Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle
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Author:
Date:
2020-04-05
[Abstract] Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have been established for the purification and characterization of exosomes. These include ultracentrifugation, ultrafiltration, size exclusion chromatography, immune capture and precipitation using a proprietary polymer. Here, we provide a protocol based on differential ultracentrifugation and sucrose density gradients tailored for the isolation of crude and ultra-pure exosomes from primary fibroblast cultures derived from adult mouse skeletal ...
[摘要] [摘要 ] 外来体是几乎所有细胞分泌的动态纳米囊泡，并存在于所有生物体液中。鉴于它们的异质含量很高，外泌体已牵涉到它们通过影响细胞-细胞和细胞-ECM通讯而发挥的许多生理和病理过程。近年来，已经建立了越来越多的方法外泌体的纯化和表征。其中包括超速离心，超滤，尺寸排阻色谱，免疫捕获和使用专有聚合物的沉淀。在这里，我们提供了基于差分超速离心和蔗糖密度梯度的协议，该协议专门用于从成年小鼠骨骼肌衍生的原代成纤维细胞培养物中分离粗制和超纯外泌体。可以对该协议进行修改和修改，以从各种组织和体液中分离和表征外泌体。
背景 ] ë xosomes是单膜，异质纳米囊泡直径范围从30至150nm，secre 由所有细胞和存在于几乎所有的体液泰德。外泌体中存在的可溶性和膜大分子，mRNA，microRNA的光谱取决于代谢状态以及分泌这些纳米囊泡的细胞的发育起源。由于它们的货物组成，外泌体可以启动接收细胞中的信号传导途径，并参与了发育，免疫和正常组织生理的维持。在神经退行性疾病，纤维化和癌症等疾病条件下，它们被证明可以触发和传播病理刺激（Rackov 等，2018; Gurunathan 等，2019; van de Vlekkert 等，2019）。在这里，我们描述了从成年小鼠腓肠肌（GA）肌肉建立的成纤维细胞培养物中纯化外泌体的方案（van de Vlekkert ...
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| Lipid Mixing Assay for Murine Myoblast Fusion and Other Slow Cell-cell Fusion Processes
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Author:
Date:
2020-03-05
[Abstract] Lipid mixing (redistribution of lipid probes between fusing membranes) has been widely used to study early stages of relatively fast viral and intracellular fusion processes that take seconds to minutes. Lipid mixing assays are especially important for identification of hemifusion intermediates operationally defined as lipid mixing without content mixing. Due to unsynchronized character and the slow rate of the differentiation processes that prime the cells for cell-cell fusion processes in myogenesis, osteoclastogenesis and placentogenesis, these fusions take days. Application of lipid mixing assays to detect early fusion intermediates in these very slow fusion processes must consider the continuous turnover of plasma membrane components and potential fusion-unrelated exchange of the ...
[摘要]
[摘要 ] 脂质混合（脂质探针在融合膜之间的分布）已广泛用于研究相对快速的病毒和细胞内融合过程的各个阶段，这些过程耗时数秒至数分钟。脂质混合测定对于鉴定在操作上定义为没有内容混合的脂质混合的半融合中间体特别重要。由于不同步的特性以及分化过程的缓慢速度，这些分化过程使细胞在成肌，破骨细胞生成和胎盘生成中引发细胞间的融合过程，因此这些融合需要几天的时间。在这些非常缓慢的融合过程中应用脂质混合测定法检测早期融合中间体时，必须考虑质膜成分的连续转换以及脂质探针在膜之间的潜在融合无关交换。在这里，我们描述了脂质混合分析在骨骼肌细胞发育和再生中成肌融合阶段的工作中的应用。我们的方法利用了基于鼠C2C12细胞的成肌分化和融合的常规体外模型。当我们观察到第一个多核细胞的外观时，我们将细胞提起并用荧光脂质DiI 作为膜探针或CellTracker TM Green 作为含量探针标记它们。通过荧光显微镜对探针在细胞之间的重新分布进行评分。半融合细胞被鉴定为用内含物和膜探针标记的单核细胞。解释必须由具有融合能力不足细胞的阴性对照系统支持，以说明脂质探针的与融合无关的交换，并将其贡献降到最低。这种方法进行了较小的修改已用于调查原代鼠成肌细胞，破骨细胞前体的融合以及由配子融合原HAP2 介导的融合，并且很可能可以用于其他缓慢的细胞-细胞融合过程。
[Bac kground ...
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| Cell Surface Protein Detection to Assess Receptor Internalization
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Author:
Date:
2016-10-20
[Abstract] The migration of membrane receptors upon exposure to different stimulants/inhibitors is of great importance. Among others, the internalization of membrane receptors affects their accessibility to ligands and cell responsiveness to environmental cues. Experimentally, receptor internalization can be used as a measure of their activation. In our studies, we employed this approach to explore cross-talk between a seven transmembrane domain receptor for neuropeptide Y (NPY), Y5R, and a tyrosine kinase receptor for brain-derived neurotrophic factor (BDNF), TrkB. To this end, we measured the internalization of Y5R upon stimulation with the TrkB ligand, BDNF. Upon treatment with BDNF, the cells were exposed to a membrane impermeable, biotinylation reagent that selectively labels surface proteins. ...
[摘要] 膜受体在暴露于不同的刺激剂/抑制剂时的迁移是非常重要的。其中，膜受体的内化影响其对配体的可及性和对环境线索的细胞应答性。在实验上，受体内化可用作其活化的量度。在我们的研究中，我们采用这种方法来探讨神经肽Y（NPY）的七个跨膜结构域受体，Y5R和脑源性神经营养因子（BDNF），TrkB的酪氨酸激酶受体之间的串扰。为此，我们测量了用TrkB配体BDNF刺激后Y5R的内化。在用BDNF处理后，将细胞暴露于选择性标记表面蛋白的不透膜的生物素化试剂。随后，生物素化的膜蛋白在具有抗生物素蛋白树脂的柱上亲和纯化，并通过蛋白质印迹分析。存在于对照和配体处理的细胞的细胞表面上的受体部分的差异用作其内化和对特定刺激的反应的量度。
[Backg 回合] 可以使用两种主要策略 - 显微镜和生物化学来测量响应外部刺激的细胞膜受体内化。最常见的方法是使用显微镜 - ...
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