| Measuring Cell Growth and Junction Development in Epithelial Cells Using Electric Cell-Substrate Impedance Sensing (ECIS)
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Author:
Date:
2020-08-20
[Abstract] Electric Cell-substrate Impedance Sensing (ECIS) is an automated method that can be used to quantify processes such as cell attachment, growth, migration and barrier functions (i.e., the properties of tight junctions). The method provides simultaneous information on cell number and tight junction function by detecting electric parameters of cells grown on electrodes. Samples are probed with small alternating current (AC) over a range of frequencies, and changes in capacitance and impedance are measured over time. Capacitance reflects the degree of electrode coverage by cells, that correlates with cell number, and can be used to assess cell proliferation or migration. Impedance values inform about barrier function. Obtaining real-time simultaneous information on these parameters ...
[摘要] [摘要 ] 电动细胞-基底阻抗感测（ECIS）是一个自动化的方法，该方法可用于定量如细胞附着，生长，迁移和屏障功能的处理（即， 紧密连接的特性）。该方法通过检测在电极上生长的细胞的电参数，提供有关细胞数量和紧密连接功能的同时信息。用小的交流电（AC）在一定频率范围内探测样品，并随时间测量电容和阻抗的变化。电容反映了细胞对电极的覆盖程度，与细胞数量相关，可用于评估细胞增殖或迁移。阻抗值告知屏障功能。获取有关这些参数的实时同步信息对于该系统而言是独一无二的，对于解决诸如紧密连接蛋白在细胞生长和迁移中的作用等基本问题具有重要价值。该协议描述了使用ECIS来跟踪肾小管上皮细胞中的细胞生长和紧密连接依赖性屏障生成。我们使用这种方法来探索如何耗尽紧密连接蛋白claudin-2如何影响肾小管细胞的生长和屏障功能。在此过程中，将细胞用对照或claudin-2特异性siRNA转染，然后在24小时后铺在电极上。随着单层细胞的发育，ECIS自动收集有关细胞生长和屏障的信息。最初使用ECIS软件对数据进行分析，然后将其导出到图形软件中以进行进一步处理。
[背景 ] 上皮细胞产生以成熟的细胞间连接为特征的单层细胞。来自连接的输入确定细胞行为，包括增殖和迁移。随着连接的发展，由于接触抑制，细胞增殖和迁移减慢，然后停止（McClatchey and ...
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| Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
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Author:
Date:
2020-07-20
[Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of claudin-2 both at the membrane and in cytoplasmic vesicles. Staining is done using a claudin-2 specific primary and a fluorescently labelled secondary antibody, along with DAPI to label nuclei. The ...
[摘要] [摘要 t] 紧密连接蛋白claudin家族的成员调节细胞旁通透性并调节细胞信号传导。在连接重塑过程中，这些蛋白被选择性地插入紧密连接或从紧密连接中检索出来，但是对这些过程的控制和协调仍不完全了解。claudins的可视化可以评估其定位和丰度的变化。我们使用所描述的方案对claudin-2染色，但它也可以用于染色任何紧密连接蛋白。我们发现使用甲醇进行固定可以使claudin-2在膜和细胞质囊泡中得到最佳保存。使用claudin-2特异性一抗和荧光标记的二抗以及与DAPI标记核一起进行染色。然后使用共聚焦显微镜对样品成像，并获得z堆栈，从而可以可视化连接和细胞内claudin-2。总的claudin-2信号可以在3D重建图像后使用Imaris 软件。
[背景 ] 紧密连接（TJ）是一种多蛋白复合物，位于连接上皮细胞的细胞间连接复合物的最顶端（Van Itallie和Anderson，2014）。这些结构产生通透性屏障和离子特异性旁细胞途径，维持apicobasal 极性，为各种重要功能提供输入并调节信号传导途径。位于TJs的蛋白可分为跨膜蛋白和相关的胞质蛋白（综述见（González-Mariscal ...
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| Metabolomic and Lipidomic Analysis of Bone Marrow Derived Macrophages
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Author:
Date:
2020-07-20
[Abstract] Macrophages are highly plastic immune cells that are capable of adopting a wide array of functional phenotypes in response to environmental stimuli. The changes in macrophage function are often supported and regulated by changes in cellular metabolism. Capturing a comprehensive picture of metabolism is vital for understanding the role of metabolic rewiring in the immune response. Here we present a method for systematically quantifying the abundance of metabolites and lipids in primary murine bone marrow derived macrophages (BMDMs). This method simultaneously extracts polar metabolites and lipids from BMDMs using a rapid two-phase extraction procedure. The polar metabolite fraction and lipid fraction are subsequently analyzed by separate liquid chromatography-mass spectrometry (LC-MS) ...
[摘要] [摘要] 巨噬细胞是高度可塑性的免疫细胞，能够对环境刺激产生广泛的功能表型。巨噬细胞功能的改变通常受到细胞代谢的支持和调节。获取新陈代谢的全面图像对于理解代谢重组在免疫反应中的作用至关重要。本文提出了一种系统定量测定小鼠骨髓源性巨噬细胞（BMDMs）中代谢物和脂质丰度的方法。该方法采用快速两相萃取法同时从BMDMs中提取极性代谢物和脂质。极性代谢物部分和脂质部分随后通过单独的液相色谱-质谱（LC-MS）方法进行分析，以优化覆盖率和定量。这使得对细胞代谢的全面表征可以用来理解各种环境刺激对巨噬细胞代谢和功能的影响。
[背景] 巨噬细胞是先天性免疫系统的细胞，在局部微环境中对信号作出反应时，可采用多种功能表型。巨噬细胞的激活与细胞代谢的特定重编程相结合并高度依赖（Tannahill et al.，2013；Galván-Peña and O'Neill，2014；Jha et al.，2015；Kelly and O'Neill，2015；Cordes et al.，2016；Mills and O'Neill 2016；等人；Mills et al.，2016；Liu et al.，2013；Lampropoulou et al.，2016；Van den ...
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