| Bone Marrow Mesenchymal Stem Cells Adhesion Assay
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Author:
Date:
2016-08-05
[Abstract] Mesenchymal stem cells (MSCs) are widespread in adult organisms and involved in tissue maintenance and repair as well as in the regulation of hematopoiesis and immunologic responses. As cell adhesion play important roles in cell interactions and signaling, to thoroughly evaluate the adhesion ability of MSCs is of vital importance to clarify the mechanism of self-renewal, proliferation, activation and migration of MSCs in different microenvironments. Based on the method by Siler et al., 2000, we revised the protocol in order to provide details on how to evaluate the adhesion ability of MSCs from bone marrow (BMSCs) on extracellular matrix (ECM) protein laminins. The current protocol can also be easily translated to MSCs with other treatments or ECMs such as collagens, fibronectin, etc ...
[摘要] 间充质干细胞（MSC）在成年生物体中广泛存在并参与组织维持和修复以及造血作用和免疫反应的调节。 由于细胞粘附在细胞相互作用和信号转导中发挥重要作用，彻底评估MSC的粘附能力对于阐明MSC在不同微环境中的自我更新，增殖，活化和迁移的机制至关重要。 基于Siler等人的方法（2000），我们修订方案以提供关于如何评价来自骨髓（BMSCs）的MSC对细胞外基质（ECM）蛋白的粘附能力的细节 层粘连蛋白。 目前的方案也可以很容易地翻译成MSC与其他治疗或ECM，如胶原，纤连蛋白，等。
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| Assessment of Mitochondrial DNA Content and Mass in Macrophages
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Author:
Date:
2016-05-05
[Abstract] Mitochondria are essential regulators in not only ATP generation and metabolic reprogramming but also the generation of reactive oxygen species (ROS) in response to pathogenic stimuli. During exposure to environmental stresses including oxidative stress, exercise, cell division and caloric restriction, mitochondria can be divided to increase mitochondrial number, size, and mass. Moreover, mitochondrial biogenesis has a crucial role in the resolution of inflammation through preserving metabolic function. Recently, diverse biochemical methods have been utilized to evaluate activity of mitochondrial biogenesis. In this protocol, we will describe an in vitro assay to measure mitochondrial DNA content and mass. Quantitative real-time PCR analysis for determination of mitochondrial DNA content ...
[摘要] 线粒体是不仅ATP生成和代谢重新编程，而且响应病原性刺激的活性氧（ROS）的生成的必要调节器。在暴露环境应激，包括氧化应激，运动，细胞分裂和热量限制期间，线粒体可以分为增加线粒体数量，大小和质量。此外，线粒体生物发生在通过保护代谢功能解决炎症中具有关键作用。最近，不同的生物化学方法已被用来评估线粒体生物发生的活性。在这个协议，我们将描述一个体外测定来测量线粒体DNA含量和质量。用于测定线粒体DNA含量的定量实时PCR分析是添加流式细胞术或共聚焦显微镜用于评价线粒体质量的有力工具。在一起，这些协议可以提供线粒体研究的重要信息。
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| Purification of Bacterial RNA from Infected Macrophages
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Author:
Date:
2015-11-20
[Abstract] Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or ...
[摘要] 在感染期间研究细菌病原体的转录组是一个非常有益的和有效的工具，用于发现有助于成功感染的基因。然而，从感染的细胞或组织中分离细菌RNA是一个挑战性的过程，因为感染细胞裂解物中宿主RNA的量高得多。我们已经优化了用于分离感染骨髓来源的巨噬细胞（BMDM）的单核细胞增生性李斯特菌（（单核细胞增生李斯特氏菌）细菌）的RNA的方法。感染后，我们裂解细胞并通过0.45μm过滤器过滤裂解物以丢弃大多数宿主蛋白和RNA。接下来，我们重新悬浮细菌，并在DNase处理后提取RNA。提取的RNA适合于通过实时PCR或微阵列的基因表达分析。我们已经在我们对感染期间的单核细胞增生李斯特氏菌基因调节的体外研究中成功地采用了该方案（Lobel等人，2015; Lobel >，2012; Kaplan Zeevi等人，2013; Rabinovich等人，2012）。
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