5-Laser Flow Cytometer

流式细胞术是一种分析单细胞的通用技术。它的基础是用一组荧光团标记细胞，然后进行流体动力学分离和激光诱导荧光分析。流式细胞术的能力来源于各种荧光标记技术，从针对细胞表面和细胞内结构(包括蛋白质和核酸)的荧光标记抗体，到摄取荧光代谢物类似物，到用于确定细胞状态的遗传编码报告，如信号通路的激活、细胞周期、细胞死亡或衰老。此外，流式细胞术非常适合于在短时间内分析复杂的细胞群体。因此，流式细胞术已经成为细胞和分子免疫学以及细胞生物学和生理学等邻近领域的一种标准方法。流式细胞术的进展迫切需要跟上对细胞类型及其特征的日益详细的描述。在这项提案中，需要一台分析型流式细胞仪，能够在5激光装置中对多达21个参数(包括19个荧光参数)进行快速和灵敏的并行分析。这种多参数5激光流式细胞仪将成为医学院流式细胞术核心设施的一部分，以及生物和化学学院分子免疫学新研究重点的一部分，以促进该大学潜在的“感染、炎症和活性物质”领域及其重点领域“RNA生物化学”的发展。它将主要用于推进旨在表征免疫细胞的发育和功能的项目。具体地说，一个项目致力于定量绘制胸腺中发育中的T细胞的细胞周期动力学图，使用细胞表面标记、DNA复制速度分析和转基因编码的荧光细胞周期指示器的组合。其他项目侧重于粘膜相关不变T细胞的动态代谢变化及其在肺生理和病理生理学中的作用，以及一般情况下的肺重建。这些项目基于对使用代谢标记的复杂亚群的分析，以及对关键信号通路的激活。其他项目侧重于在T细胞受体依赖的信号通路的背景下分析microRNA的活性，描述传染病以及多发性硬化症相关病理中的免疫细胞亚群的特征。将所要求的多参数5激光流式细胞仪行政整合到一个核心设施结构中，将确保其高效和跨学科使用。

Flow cytometry constitutes a versatile technology for the analysis of single cells. It is based on labeling of cells with a combination of fluorophores followed by hydrodynamic separation and analysis of laser-induced fluorescence. The power of flow cytometry derives from a large variety of fluorescent labeling techniques, ranging from fluorochrome-conjugated antibodies directed against cellular surface as well as intracellular structures, including proteins and nucleic acids, via uptake of fluorescent metabolite analogs to genetically encoded reporters for defined cellular states, such as activation of signaling pathways, cell cycle, cell death or senescence. Furthermore, flow cytometry is ideally suited to analyze complex cell populations in a short amount of time. Accordingly, flow cytometry has emerged as a standard method in cellular and molecular Immunology as well as in adjacent fields like Cell Biology and Physiology. Progress in flow cytometry critically needs to keep track with the increasingly detailed description of cell types and their characteristics. In this proposal, an analytical flow cytometer is requested, capable of rapid and sensitive parallel analysis of up to 21 parameters (including 19 fluorescent parameters) in a 5-laser setup. This multiparameter 5-laser flow cytometer will become part of the flow cytometry core facility of the Faculty of Medicine as well as part of a new research focus of Molecular Immunology of the Faculty of Biology and Chemistry to foster the university’s area of potential “Infections, Inflammation and Active Substances” and its accent area “RNA Biochemistry”. It will primarily serve to advance projects aimed at characterizing development and function of immune cells. Specifically, one project is dedicated to quantitatively map cell cycle dynamics of developing T cells in the thymus, employing combinations of cell surface markers, analysis of DNA replication speed and transgenically encoded fluorescent cell cycle indicators. Additional projects focus on the dynamic metabolic changes in Mucosa-associated invariant T cells and their function in physiology and pathophysiology of the lung as well as lung remodeling upon insult in general. These projects are based on the analysis of complex subpopulations employing metabolic markers as well as the activation of critical signaling pathways. Further projects focus on the analysis of microRNA activity in the context of T cell receptor-dependent signaling pathways, characterization of immune cell subpopulations in the epidydimis as well as in multiple-sclerosis-related pathology. Administrative integration of the requested multiparameter 5-laser flow cytometer into a core facility structure will ensure its efficient and interdisciplinary use.