5-Laser Spectral Flow Cytometer

我们应用光谱流式细胞仪对免疫细胞和细胞外小泡进行高参数分析。这里应用的光谱流式细胞仪是为了补充分子免疫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肝脏等外围器官分离的免疫细胞群是必不可少的，在这些器官中，它们获得了非常高的自发荧光，这可以通过使用光谱流式细胞术来最好地补偿。此外，在肝脏或癌组织慢性病毒感染的背景下研究稀有免疫细胞群，如抗原特异性CD8T细胞，需要高度的参数分析，以实现对少数可供分析的细胞的深入表型和功能表征。在过去的几年里，我们使用光谱流式细胞术来鉴定(1)一种由髓系来源的抑制细胞产生的新的抑制代谢物，它导致肿瘤组织内T细胞的完全代谢关闭(Baumann等人)。自然免疫学2020)，(2)通过嘌呤能受体诱导T细胞激活的一种新形式，其决定脂肪肝的肝脏病理，(3)肠道中树突状细胞在介导炎症和引起炎症性肠病中的作用，(4)迄今未知的树突状细胞亚群，其激活肿瘤间质内的T细胞以引发保护性抗癌免疫，(5)关闭T细胞激活的肝组织变阻器，以及(6)基于光谱的流式细胞术开发如何分析单个外切体的技术，它允许追踪单个细胞的外切体，如癌细胞和患者体内感染SARS-CoV-2的细胞。为了扩大我们的能力，我们计划使用这里应用的光谱流式细胞仪，使用光谱流式细胞仪对来自外周组织的稀有免疫细胞群进行高参数表型和功能表征。迫切需要第二台光谱流式细胞仪，因为分子免疫研究所现有的光谱流式细胞仪是在6年多前获得的，不再满足该研究所在第三方资助项目中工作的科学家的要求，尽管这种仪器每天从早上8点到晚上10点都在使用此外，在临床前疾病模型和患者血液中，应用光谱流式细胞仪表征小泡的能力的提高将是表征单个外切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.