| Coculture between hMADS and Mouse Adult CM
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Author:
Date:
2014-07-20
[Abstract] Heart failure occurring after acute myocardial infarction (MI) is among the main causes of death in western countries. Cell therapies, particularly those based on mesenchymal stem cells (MSC), represent one of the most promising approaches to repair damaged heart tissues. Several reports have provided evidences that injection of mesenchymal stem cells improved heart function following myocardial infarction (Shake et al., 2002; Zimmet and Hare, 2005; Zeng et al., 2007). Nevertheless, the mechanism(s) by which MSC exert their therapeutic action is far from being understood, and further knowledges in this field are required especially to optimize efficiency of current cardiac cell therapies. To assess the regenerative mechanisms developed by MSC in vitro, we ...
[摘要] 急性心肌梗死（MI）后发生的心力衰竭是西方国家死亡的主要原因。细胞疗法，特别是基于间充质干细胞（MSC）的那些，代表了修复受损心脏组织的最有希望的方法之一。几个报道提供了证据，注射间充质干细胞改善了心肌梗塞后的心脏功能（Shake等人，2002; Zimmet和Hare，2005; Zeng等人， 2007）。然而，MSC发挥其治疗作用的机制远未被理解，并且尤其需要在该领域中的进一步知识以优化当前心脏细胞治疗的效率。为了评估由MSC在体外形成的再生机制，我们开发了上述方法，其预期模拟梗塞心脏的典型微环境。该方法包括处于不良状态的小鼠终末分化心肌细胞与本文用作MSC模型的人多能脂肪来源干细胞（hMADS细胞）之间的物种错配共培养物。
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| Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation
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Author:
Date:
2014-04-05
[Abstract] The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become LC3-PE or LC3-II predominantly localised on the autophagosomal membranes (outer and inner). Autolysosomal content of LC3-II is very low as upon autophago/lysosomal fusion it is either cleaved off from the outer membrane by Atg4 or degraded together with the inner membrane by the lysosomal activity. Therefore GFP-LC3 and mCherry-GFP-LC3 might be visualized by conventional or confocal fluorescence microscopy (FM). In this situation mCherry-GFP-LC3 or ...
[摘要] 自噬蛋白，LC3代表自噬体结构的可靠特征标记。最初的LC3由半胱氨酸蛋白酶自噬相关基因4（Atg4）在其C末端处理，以产生通常位于细胞质中的LC3-I。之后LC3-I与磷脂酰乙醇胺（PE）缀合以变成主要定位在自噬体膜（外部和内部）上的LC3-PE或LC3-II。 LC3-II的自溶酶体内含物非常低，因为在自噬/溶酶体融合时，其被Atg4从外膜切割或与内膜一起通过溶酶体活性降解。因此，GFP-LC3和mCherry-GFP-LC3可能通过常规或共聚焦荧光显微镜（FM）可视化。在这种情况下，mCherry-GFP-LC3或GFP-LC3细胞质池被可视化为均匀分散的信号，并且含有mCherry-GFP-LC3-II或GFP-LC3-II的自噬体被检测为点状形成。斑点数可以用作自噬体丰度的标志物。一般来说，我们建议计数每个细胞的GFP-LC3斑点的平均数。
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| Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells
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Author:
Date:
2014-04-05
[Abstract] Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the autophagosomal content might be. The method is based on Cyto-ID staining of autophagic compartments (pre-autophagosomes, autophagosomes, and autophagolysosomes) in live cells using Cyto-ID® Autophagy Detection Kit. Autophagic compartments are intermediate constituents of a dynamic lysosomal degradation process and their intracellular abundance at a particular time point is a function of the established equilibrium between their ...
[摘要] 流式细胞术允许非常灵敏和可靠的高通量分析自噬通量。该方法允许在流动中筛选细胞并捕获多组分图像。使用这种技术，可以在悬浮液以及粘附细胞中在胰蛋白酶消化时精确分析自噬流，而与自噬体内容物的异质性无关。该方法基于使用Cyto-ID自动吞噬检测试剂盒的活细胞中的自体吞噬细胞（前自噬体，自噬体和自噬溶酶体）的Cyto-ID染色。自噬区室是动态溶酶体降解过程的中间组分，并且它们在特定时间点的细胞内丰度是其产生和降解之间建立的平衡的函数。自噬吞噬的确定有助于自噬体形成的早期诱导和自噬体成熟的晚期抑制之间的区分，因为两者都导致自噬体存在的最终增加。 Cyto-ID测定基于使用选择性染色自噬隔室的特定染料，因此允许测定自噬通量，作为染色隔室在碱性或活化条件[雷帕霉素（1-5μmol/L），PP242（1- （NH 4 Cl）（10-20μmol/L）或含有6mmol/L葡萄糖（饥饿培养基）的Hanks平衡盐溶液），使用溶酶体化合物阻断自噬溶酶体降解mmol/L）或氯喹（CQ）（5-10μmol/L）。 ΔMFICyto-ID = MFI Cyto-ID（+ CQ/NH 4 Cl）-MFI Cyto-ID（-CQ/NH 4 Cl）。
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