5-Laser Spectral Flow Cytometer

我们申请光谱流式细胞仪进行免疫细胞和细胞外囊泡的高参数分析。本文所用的光谱流式细胞仪旨在补充分子免疫学研究所现有的流式细胞仪，并满足科学家使用光谱流式细胞仪进行实验的需要。在我们过去几年的实验中，光谱流式细胞术对于分析从外周器官（如肝脏）分离的免疫细胞群是必不可少的，在这些器官中，它们获得非常高的自体荧光，这可以通过使用光谱流式细胞术得到最好的补偿。此外，研究罕见的免疫细胞群体，如在肝脏慢性病毒感染或癌组织中的抗原特异性CD 8 T细胞，需要高参数分析，以实现可用于分析的少数细胞的深入表型和功能表征。在过去的几年里，我们已经使用光谱流式细胞术来鉴定（1）髓源性抑制细胞产生的一种新的抑制性代谢物，其导致肿瘤组织内T细胞的代谢完全关闭（Baumann等人，Nature Immunology 2020），（2）通过嘌呤能受体的代谢物诱导的T细胞活化的新形式，其决定脂肪肝疾病中的肝脏病理，（3）树突状细胞在肠道中介导炎症并引起炎性肠病的作用，（4）迄今未知的树突状细胞亚群，其激活肿瘤基质内的T细胞以引发保护性抗癌免疫，（5）肝组织变阻器，其关闭T细胞的激活，以及（6）开发基于光谱流式细胞术的技术，如何分析单个外来体，其允许追踪来自个体细胞如癌细胞和患者中的SARS-Cov-2感染细胞的外来体。计划使用此处应用的光谱流式细胞仪，以扩大我们使用光谱流式细胞术对外周组织中罕见免疫细胞群进行高参数表型和功能表征的能力。迫切需要第二台光谱流式细胞仪，因为分子免疫学研究所现有的光谱流式细胞仪已经在6年多前获得，不再满足研究所从事第三方资助项目的科学家的需求，尽管该仪器每天上午8点至晚上10点使用。此外，用于表征小尺寸囊泡的光谱流式细胞仪的能力增加将是表征临床前疾病模型和患者血液中单个外泌体及其表面表型的关键。

We apply for a spectral flow cytometer to perform high-parametric analysis of immune cells and extracellular vesicles. The spectral flow cytometer applied for here is intended to complement already existing flow cytometers at the Institute of Molecular Immunology and to meet the need of the scientists to perform experiments with a spectral flow cytometer. Spectral flow cytometry in our experiments over the last years was indispensable for analysis of immune cell populations isolated from peripheral organs such as the liver where they acquire a very high autofluorescence, which can be best compensated for by using spectral flow cytometry. Furthermore, studying rare immune cell populations such as antigen-specific CD8 T cells in the context of chronic viral infection of the liver or in cancer tissue requires high parametric analysis to achieve in depth phenotypic and functional characterization of the few cells available for analysis. Over the last years, we have used spectral flow cytometry to identify (1) a novel inhibitory metabolite produced by myeloid-derived suppressor cells that leads to complete metabolic shut-off of T cells within tumor tissue (Baumann et al. Nature Immunology 2020), (2) a novel form of metabolite-induced T cell activation through purinergic receptors that determines liver pathology in fatty liver disease, (3) a role of dendritic cells in the gut to mediate inflammation and cause inflammatory bowel disease, (4) a so far unknown dendritic cell subpopulation that activates T cells within tumor stroma to elicit protective anti-cancer immunity, (5) a liver tissue rheostat that shuts off T cells from activation and (6) to develop a technique based on spectral flow cytometry how to analyse single exosomes, which allows to trace exosomes from individual cells such as cancer cells and SARS-Cov-2 infected cells in patients. It is planned to use the spectral flow cytometer applied for here for the expansion of our capacity to use spectral flow cytometry to perform high-parametric phenotypic and functional characterization of rare immune cell populations from peripheral tissues. A second spectral flow cytometer is urgently needed because the existing spectral flow cytometer at the Institute of Molecular Immunology, that has been obtained more than 6 years ago, does not meet any more the demands from scientists at the institute working in third-party funded projects, although this instrument is used every day from 8 am to 10 pm. Furthermore, the increased capacity of the spectral flow cytometer applied for here to characterize small-sized vesicles will be key to characterize single exosomes and their surface phenotype in preclinical disease models and patient blood.