| Mouse Embryonic Fibroblast Cell Culture and Stimulation
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Author:
Date:
2016-07-05
[Abstract] Culture of mouse embryonic fibroblast (MEF) cells represents a powerful system to test gene function due to their easy accessibility, rapid growth rates, and the possibility of a large number of experiments. Fibroblasts are a group of heterogeneous resident cells of mesenchymal origin that have various locations, diverse appearances and distinctive activities. Because of their ubiquitous distribution as tissue cells, these cells are poised to respond to factors released by newly activated innate immune cells, thus becoming a useful tool to study inflammation and immunity. Here, we describe procedures for mouse embryonic fibroblast cell isolation, primary culture, and stimulation. Specifically, we have optimized a step of serum starvation prior to stimulation. This step is necessary to ...
[摘要] 小鼠胚胎成纤维细胞（MEF）细胞的培养代表了测试基因功能的强大系统，因为它们容易获得，快速生长速率和大量实验的可能性。成纤维细胞是一组具有不同位置，不同外观和独特活动的间充质来源的异质驻留细胞。由于它们作为组织细胞的普遍分布，这些细胞准备响应由新激活的先天免疫细胞释放的因子，因此成为研究炎症和免疫的有用工具。在这里，我们描述了小鼠胚胎成纤维细胞分离，原代培养和刺激的程序。具体来说，我们已经优化了刺激前血清饥饿的步骤。这个步骤是必要的，以维持这些细胞在它们暴露于促炎刺激为最佳反应之前的静止状态。如在我们以前的研究中所示，这些小鼠成纤维细胞在通过常规Northern印迹技术容易检测的水平下不表达Tnf ， Csf2 或 Lai WS等人，2006）。
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| Mitochondrial Transmembrane Potential (ψm) Assay Using TMRM
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Author:
Date:
2013-12-05
[Abstract] During cellular respiration, nutrients are oxidized to generate energy through a mechanism called oxidative phosphorylation, which occurs in the mitochondria. During oxidative phosphorylation, the gradual degradation of molecules through the TCA cycle releases electrons from the covalent bonds that are broken. These electrons are captured by NAD+ through its reduction into NADH. Finally, NADH transports the electrons to the complexes of the electron chain in the internal membrane of mitochondria. These complexes use the energy released by the electrons to pump protons into the intermembrane space, generating an electrochemical gradient across the internal membrane of mitochondria, which provides energy for the ATP-synthase complex, ultimately producing adenosine triphosphate (ATP). We ...
[摘要] 在细胞呼吸期间，营养物通过称为氧化磷酸化的机制被氧化以产生能量，所述机制发生在线粒体中。在氧化磷酸化过程中，分子通过TCA循环的逐渐降解从被破坏的共价键释放电子。这些电子被NAD +捕获，通过其还原成NADH。最后，NADH将电子传输到线粒体内膜中的电子链的复合物。这些复合物使用由电子释放的能量将质子泵入膜间空间，产生穿过线粒体内膜的电化学梯度，其为ATP-合酶复合物提供能量，最终产生三磷酸腺苷（ATP）。我们使用四甲基罗丹明甲酯（TMRM），一种细胞渗透性，阳离子，红色荧光染料评估线粒体膜电位（ψsub）。为了特异性地测量线粒体膜电位（ψm），我们定量了应用FCCP（线粒体电子链解偶联）之前和之后的荧光强度。应用FCCP之前和之后的强度差异具体对应于线粒体膜电位。我们通过细胞荧光法分析线粒体膜电位（ψm）。信号的总电平与解耦之后产生的信号之间的比率提供了对于单元大小差异的归一化值。此外，为了归一化我们分析的不同大小的细胞，我们已经使用IN细胞分析仪分析了活体成像中的TMRM，使得可以测量每单位线粒体膜面积的线粒体膜电位水平。因此，我们的协议也可以用于比较不同大小的细胞的线粒体膜电位。
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