| DNA Damage Induction by Laser Microirradiation
|
|
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 ...
|
|
|
| Gentamicin Protection Assay to Determine Bacterial Survival within Macrophages
|
|
Author:
Date:
2014-09-20
[Abstract] Macrophages are key cells involved in orchestrating host defense against infections. Here, we describe the protocol for a bacterial killing assay in macrophages that can be adapted to any bacterial pathogen. Using this assay, we analyzed the survival of wild-type and mutant strains of Escherichia coli (E. coli) within RAW 264.7 cells, a widely used macrophage cell line. Bacterial mutants defective in intracellular survival within macrophages can be delineated using this assay.
[摘要] 巨噬细胞是参与编制宿主防御感染的关键细胞。 在这里，我们描述的细菌杀伤测定在巨噬细胞，可以适应任何细菌病原体的协议。 使用该测定，我们分析了广泛使用的巨噬细胞系RAW 264.7细胞内的大肠杆菌（大肠杆菌）的野生型和突变株的存活。 可以使用该测定来描绘巨噬细胞内的细胞内存活缺陷的细菌突变体。
|
|
|