| Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs
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Author:
Date:
2016-03-05
[Abstract] Numerous biological functions such as cytokinesis, changes in cell shape and cell migration require actomyosin-driven cellular contractility. However, the detailed mechanism of how contractile forces drive cellular processes are difficult to decipher due to the complexity of the intracellular environment. In particular, the mesoscopic description of the myosin II-dependent actin retrograde flow in cell lamellum is missing. Here, we describe a methodology for detergent extraction of cell, which preserves integrity of the actin cytoskeleton. This semi-in vitro cell model allows for the observation, using light microscopy, and quantification of changes in the actin cytoskeleton resulting from the activation of cellular contractility upon addition of ATP. This assay also allows for ...
[摘要] 许多生物学功能如细胞分裂，细胞形状和细胞迁移的变化需要肌动蛋白驱动的细胞收缩性。然而，由于细胞内环境的复杂性，收缩力如何驱动细胞过程的详细机制难以解读。特别是，细胞层中的肌球蛋白II依赖性肌动蛋白逆行流的介观描述缺失。在这里，我们描述了洗涤剂提取的细胞，保留肌动蛋白细胞骨架的完整性的方法。这种半体外细胞模型允许使用光学显微镜观察和定量在添加ATP时由于细胞收缩性的活化而导致的肌动蛋白细胞骨架中的变化。该测定还允许评估肌动蛋白相关蛋白和其他相关因子在肌动蛋白收缩活性的调节中的作用。在这里，我们证明逆行流动的着名的基于肌动蛋白的结构 - 横向弧，这是肌球蛋白IIA含有结构出现在lamellipodium-lamellum之间的边界和肌球蛋白II依赖的方式向心移动。
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| Nanofluidic Proteomic Immunoassay
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Author:
Date:
2015-07-20
[Abstract] Nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, has the potential to quantitatively characterize protein post-translational modifications that cause shifts in isoelectric point (pI). This protocol details the NIA analysis of protein phosphorylation using AKT as an example.
This protocol can be used for two platforms, NanoPro 1000 and Peggy Sue, from ProteinSimple Company. NanoPro 1000 separates proteins based on charge. Peggy Sue separates proteins based on either charge or size. The platforms can analyze up to 96 samples at a time with lysates from as few as 25 cells per assay. Detailed information of the platforms is available on ProteinSimple’s website (http://www.proteinsimple.com ...
[摘要] 纳米流体蛋白质组学免疫测定（NIA），由等电聚焦跟随敏感化学发光检测组成，具有定量表征导致等电点（pI）移位的蛋白质翻译后修饰的潜力。 该协议详细描述了使用AKT作为实例的蛋白质磷酸化的NIA分析。
此协议可用于ProteinSimple公司的两个平台NanoPro 1000和Peggy Sue。 NanoPro 1000基于电荷分离蛋白质。 Peggy Sue基于电荷或大小分离蛋白质。 平台可以一次分析高达96个样品，每个测定每裂解物少至25个细胞。 有关平台的详细信息，请访问ProteinSimple的网站（ http://www.proteinsimple.com ）。
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| In vitro Regulatory T cells Differentiation From Naïve T Cells
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Author:
Date:
2014-03-20
[Abstract] In the past years, a subset of regulatory T cells (Tregs) expressing CD4, CD25 and the transcription factor FoxP3 has gained considerable attention as key regulators of T-cell tolerance and homeostasis (Sakaguchi, 2004). This population of T cells is specifically engaged in the maintenance of immune self-tolerance and the control of aberrant immune responses to foreign antigens. Remarkably, regulatory T cells have been implicated in tumor cell evasion of immune responses (Curiel et al., 2004; Zou, 2006) by suppressing T cell mediated antitumor immunity. The study of the signals that promote the differentiation of this suppressive population in the tumor microenvironment has become a central issue. Here we described a detailed method to in vitro differentiate Tregs using ...
[摘要] 在过去的几年中，表达CD4，CD25和转录因子FoxP3的调节性T细胞（Treg）的一个子集已经作为T细胞耐受性和体内平衡的关键调节剂获得了相当大的关注（Sakaguchi，2004）。 这种T细胞群特别参与维持免疫自身耐受性和控制对外来抗原的异常免疫应答。 值得注意的是，调节性T细胞通过抑制T细胞介导的抗肿瘤免疫参与了肿瘤细胞逃避免疫应答（Curiel等人，2004; Zou，2006）。 促进这种抑制群体在肿瘤微环境中的分化的信号的研究已经成为一个中心问题。 在这里，我们描述了使用来自小鼠天然T细胞的肿瘤细胞条件培养基来体外鉴别Treg的详细方法，并且基于它们的特异性标记物来鉴定它们（Dalotto-Moreno等人， >，2013）。
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