| Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes
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Author:
Date:
2017-05-05
[Abstract] Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are ...
[摘要] 单核细胞增生利斯特氏菌是一种重要的革兰氏阳性食源性病原体，是即食食品中的一个特殊问题。它具有在诸如制冷温度和高盐浓度的恶劣条件下生存的能力，并且已知可以穿过肠，胎盘和血脑屏障。已经使用了诸如子宫颈，肝脏，树突状细胞，肠和巨噬细胞的几种癌细胞系来研究人细胞中李斯特菌的体外扩增和存活。人肠上皮细胞已被用于研究李斯特菌如何穿过肠屏障并引起感染。本文中的方案描述了生长Caco-2细胞的过程，维持细胞并将其用于粘附和侵袭测定。在粘附测定期间，将细胞与李斯特菌孵育30分钟，但是在侵袭测定中，细胞生长在感染后的几个时间点被停止以监测细胞中李斯特菌的生长和存活率。
背景 ...
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| DNA Damage Induction by Laser Microirradiation
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Author:
Date:
2016-12-05
[Abstract] Genome instability can lead to cell death, senescence and cancerous transformation. Specific repair pathways have evolved to prevent accumulation of DNA lesions. Studying these highly dynamic and specific repair pathways requires precise spatial and temporal resolution, which can be achieved through a combination of laser microirradiaiton and live cell microscopy. DNA lesions are introduced at pre-determined sub-nuclear sites and repair can be analyzed in real time in living cells when using fluorescently tagged repair proteins (Mortusewicz et al., 2008). Alternatively, laser microirradiation can be combined with immunofluorescence analysis to study recruitment of endogenous proteins to laser-induced DNA damage tracks that can be visualized by positive controls like, e.g., ...
[摘要] 基因组不稳定性可导致细胞死亡，衰老和癌性转化。特异性修复途径已经进化以防止DNA损伤的累积。研究这些高度动态和特定的修复途径需要精确的空间和时间分辨率，这可以通过激光微激光和活细胞显微镜的组合实现。当使用荧光标记的修复蛋白时，在预定的亚核位点引入DNA损伤并且可以在活细胞中实时分析修复（Mortusewicz等人，2008）。或者，激光微辐照可与免疫荧光分析结合以研究内源蛋白质对激光诱导的DNA损伤轨迹的募集，其可通过阳性对照例如标记DNA断裂位点的γH2AX显现。
关键字：微辐射，活细胞成像，DNA损伤，DNA修复，DNA损伤，DNA损伤反应，免疫荧光，显微镜等
/strong>哺乳动物细胞的基因组完整性不断受到通过外部和内部来源引入的DNA损伤的挑战。最常见的DNA损伤是氧化碱基，双链断裂，单链断裂，链间和链内交联和UV加合物。已经发展了各种DNA损伤信号传导和修复途径以处理这些损伤。为了使DNA修复快速，精确和有效，涉及感测，信号传导和修复特定DNA损伤的许多蛋白质必须在空间和时间上协调。此外，DNA被组织成更高级的染色质结构，因此对于DNA损伤，DNA修复酶是可及的，染色质必须重塑。激光微照射与高级活细胞显微镜相结合允许在活细胞的上下文中研究这些高度动态的过程（Mortusewicz等人，2008）。这里描述的协议使用405 ...
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| Sample Preparation and Fractionation of Arabidopsis thaliana Sperm and Vegetative Cell Nuclei by FACS
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Author:
Date:
2015-11-20
[Abstract] One of the major topics in plant and animal biology is sexual reproduction. It is, therefore, of great interest to isolate and study germ cells and accessory cells. The male gametophyte of the flowering plant Arabidopsis thaliana (A. thaliana), pollen, is the product of two post-meiotic mitotic divisions. Each mature pollen grain consists of two sperm cells contained within the vegetative cell, the non-reproductive companion cell. The tough pollen wall and its special nested structure make it difficult to study pollen cells separately. Here, we describe a simple and efficient method to fractionate A. thaliana sperm and vegetative cell nuclei by fluorescence activated cell sorting (FACS). Our protocol is based on differences in fluorescence intensity of sperm and ...
[摘要] 植物和动物生物学的一个主要议题是有性繁殖。因此，分离和研究生殖细胞和附属细胞是非常有意义的。开花植物拟南芥（Arabidopsis thaliana）（ A。thaliana ）的雄性配子体是两个减数分裂后有丝分裂的产物。每个成熟花粉粒由包含在营养细胞（非生殖伴侣细胞）内的两个精细胞组成。坚硬的花粉壁及其特殊的嵌套结构使得难以分别研究花粉细胞。在这里，我们描述一个简单而有效的方法分割。拟南芥精子和营养细胞核通过荧光激活细胞分选（FACS）。我们的方案基于用SYBR Green I染色的精子和营养细胞核的荧光强度的差异.100植物产生约1×10 6个精子和350,000个营养细胞核。该方法可用于纯化各种A的花粉核。拟南芥野生型种质和突变株系，并且原则上可适用于其它植物物种的花粉。
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