Neutrophil Microscopy and Quantitative Imaging Core B

摘要 中性粒细胞是抵御细菌和真菌的第一道防线，并引发炎症。对中国传统文化的研究 因此，调节中性粒细胞活化的分子机制具有重要意义。将这些可视化 机制需要独特的、最先进的技术和方法。为了实现这一点，核心B被设计为 协调、设计、开发和执行用于理解中性粒细胞的新技术方法 炎症中的生物学。核心B将与所有三个项目合作，并将提供相关的成像和显微镜 在小鼠和人类样本中进行检测。核心利用可用的尖端技术来实施新的 实验方法和帮助阐明研究计划中提出的复杂的细胞生物学过程 项目1(Hedrick博士)、项目2(Catz博士)和项目3(Hoffman博士)。为此，我们将拥有 技术核心总监(Kiosses博士)的专注工作，他将分配具体的时间和精力来支持 PPG的目标是。核心B优化了中性粒细胞成像、图像量化和数据的工作流程 共享所有项目。它提供了严格的质量控制，并确保了多个 应用于研究中性粒细胞和前驱细胞在冠状动脉疾病中的关键技术。 核心B将实施独特的活细胞方法来研究中性粒细胞泡运输，细胞骨架 重塑和细胞死亡途径。核心B在技术和概念上都是创新的，因为它利用了， 中性粒细胞专用的新方法，包括超分辨率显微镜和定量分析 中性粒细胞动力学，以阐明中性粒细胞的功能及其在炎症中的作用。在关系中 在项目1中，核心B将在NETsis期间识别动脉粥样硬化斑块中的中性粒细胞及其前体 以及在高脂饮食斑块发展的不同阶段炎性小体激活的条件下的下睑下垂， 体外和体内使用载脂蛋白E-/-西方饮食模型的CAD小鼠。与项目2，核心 B将利用单分子超分辨率显微镜和全内反射荧光显微镜 研究中性粒细胞前体细胞囊泡转运和肌动蛋白重塑的分子机制。 B核心还将利用相关光电子显微镜(Clem)来表征新发现的 前体特异性颗粒(PSG)。在与项目3的合作中，核心B将检查三维 细胞发生凋亡或坏死时线粒体网络的定位和空间变化 小鼠模型的中性粒细胞及其前体细胞和患者的人中性粒细胞的死亡 CAD。总而言之，Core B的独特设计是为了最大限度地利用La Jolla提供的宝贵技术 尽可能最大限度地提高与PPG相关的中性粒细胞的研究和生产率。

SUMMARY Neutrophils are the first line of defense against bacteria and fungi and induce inflammation. The study of the molecular mechanisms mediating neutrophil activation is therefore of fundamental importance. Visualizing these mechanisms requires unique, state-of-the-art technologies and approaches. To achieve this, Core B is designed to coordinate, design, develop and execute new technical approaches applied to the understanding of neutrophil biology in inflammation. Core B will work with all three Projects and will provide relevant imaging and microscopy assays in mice and human samples. The core utilizes available cutting-edge technology to implement new experimental approaches and to help elucidate the intricate cell biology processes proposed in research plans of Projects 1 (Dr. Hedrick), Project 2 (Dr. Catz) and Project 3 (Dr. Hoffman). To this end, we will have the dedicated effort of a Technology Core director (Dr. Kiosses) who will allocate specific time and efforts to support the goals of the PPG. Core B optimizes the workflows of neutrophil imaging, image quantification and data sharing for all projects. It provides stringent quality controls and ensures uniformity of performance of multiple key technologies applied to the study of neutrophils and precursors in the setting of coronary artery disease. Core B will implement unique live-cell approaches to study neutrophil vesicular trafficking, cytoskeleton remodeling and cell death pathways. Core B is technically and conceptually innovative because it utilizes, neutrophil-dedicated, novel approaches that include super-resolution microscopy and quantitative analyses of neutrophil dynamics to elucidate neutrophil functions and to elucidate their role in inflammation. In relationship with Project 1, Core B will identify neutrophils and their precursors in atherosclerotic plaques during NETosis and pyroptosis in conditions of inflammasome activation at various stages of plaque development in high-fat diet, both ex-vivo and in vivo using the ApoE-/- Western diet model of CAD mice. In association with Project 2, Core B will utilize single-molecule super-resolution microscopy and Total Internal Reflection Fluorescence Microscopy to study molecular mechanisms regulating vesicular trafficking and actin remodeling in neutrophil precursors. Core B will also utilize Correlated-Light Electron Microscopy (CLEM) to characterize the newly identified precursor-specific granules (PSGs). In collaboration with Project 3, Core B will examine three-dimensional localization and spatial changes in the mitochondrial network as the cells undergo apoptotic or necroptotic cell death in both neutrophils and their precursors from mouse models and human neutrophils from patients with CAD. In conclusion, Core B is uniquely designed to use the valuable technology available in La Jolla to the fullest extent possible to maximize neutrophil-dedicate research and productivity associated with the PPG.