| Carboxyfluorescein Dye Uptake to Measure Connexin-mediated Hemichannel Activity in Cultured Cells
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Author:
Date:
2021-02-05
[Abstract] Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased ...
[摘要] [摘要]连接蛋白是有助于直接和局部旁分泌通过自己的能力介导的细胞-细胞通信膜结合蛋白寡聚成六聚半通道。当相邻通道对齐时，它们形成间隙连接，为单元之间的信息传输提供直接路径。与通常在生理条件下打开的完整的间隙连接相反，未对接的半通道的打开概率较低，主要是在受伤时打开。半通道允许小分子和离子（约1kDa）释放到局部细胞间环境中，并且过度表达/活性 与许多疾病状况有关。羧基荧光素染料的摄取量度了半通道的功能性表达，其中增加的半通道活性/功能反映了增加的负荷。该技术依赖于通过开放性半通道对不可渗透膜的荧光示踪剂的吸收，可用于比较在不同条件下（例如对照与疾病）培养的细胞单层之间的通道活性。其他技术（例如生物素化和电生理学）可以分别测量细胞表面表达和半通道开放可能性，但是，羧基荧光素的摄取提供了一种简单，快速且经济高效的方法来测定多种细胞类型的体外半通道活性。
图形摘要：
使用染料吸收量度半通道活性
[背景]连接蛋白（CX ）是一体的跨膜蛋白，其寡聚到连接子在细胞表面。连接子与相似的六聚体蛋白复合物停靠在相邻细胞上，形成用于间隙连接细胞间通讯的双向导管（GJIC；Bosco等人，2011 ...
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| Quantitative 3D Time Lapse Imaging of Muscle Progenitors in Skeletal Muscle of Live Mice
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Author:
Date:
2016-12-20
[Abstract] For non-optically clear mammalian tissues, it is now possible to use multi-photon microscopy to penetrate deep into the tissue and obtain detailed single cell images in a live animal, i.e., intravital imaging. This technique is in principle applicable to any fluorescently marked cell, and we have employed it to observe stem cells during the regenerative process. Stem cell-mediated skeletal muscle regeneration in the mouse model has been classically studied at specific time points by sacrificing the animal and harvesting the muscle tissue for downstream analyses. A method for direct visualization of muscle stem cells to gain real-time information over a long period in a live mammal has been lacking. Here we describe a step-by-step protocol adapted from Webster et al. ...
[摘要] 对于非光学清晰的哺乳动物组织，现在可以使用多光子显微镜深入渗透到组织中，并且在活体动物（即，即活体成像）中获得详细的单细胞图像。这种技术原则上适用于任何荧光标记的细胞，我们已经使用它来观察再生过程中的干细胞。小鼠模型中的干细胞介导的骨骼肌再生已经在特定时间点通过牺牲动物和收获肌肉组织进行下游分析进行了经典研究。一直缺乏直接观察肌肉干细胞以在活体哺乳动物中长时间获得实时信息的方法。在这里，我们将介绍一种从Webster等人修改的分步骤协议。 （2016），以定量测量稳态期间荧光标记（GFP，EYFP）肌肉干细胞和祖细胞的行为以及肌肉损伤后的行为。
背景干细胞和祖细胞的长期体内成像首先在无外科手术的连续生理再生中用于毛囊（Rompolas等人，2012）。相比之下，骨骼肌的干细胞在正常的内稳态期间主要是静止的和无活性的。需要肌肉的损伤来激活肌肉干细胞来安装再生过程。肌肉干细胞/祖细胞的体外成像已被广泛用于人造环境中的研究。为了了解在自然环境中再生过程中的肌肉干细胞行为，我们开发了一种在骨骼肌再生过程中对其进行成像的方法。我们的方法允许高达8小时的连续成像每次每次伤害后。这是在受伤/再生环境（Webster等人，2016）中首次观察到体内骨骼肌干细胞。
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