Five-laser Aurora spectral flow cytometer

五激光 Aurora 光谱流式细胞仪

• 批准号: 10426837
• 负责人: Remi J Creusot
• 金额: $ 36.08万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10426837
• 关键词: Biomedical Research; Biopsy; Blood; Cells; Color; Cytometry; Detection; Flow Cytometry; Fluorochrome; Funding; Human; Incentives; Lasers; Life; Maintenance; Masks; Measures; Medical center; Organoids; Patients; Principal Investigator; Proteins; Reagent; Reporter; Research Personnel; Resolution; Sampling; Services; Side; Signal Transduction; Surface; Technology; Testing; Tissues; United States National Institutes of Health; Universities; Viola; Work; cell type; cost; design; detector; flexibility; humanized mouse; instrument; lymph nodes; operation; optical spectra; particle; single cell analysis; tool

PROJECT SUMMARY Flow cytometry is an essential tool for the single cell analysis of a wide variety of cell types to assess the expression of surface and intracellular markers and perform functional measures. Conventional flow cytometry has been unable to leverage the large variety of fluorochromes available for marker analysis, because the technology is focused on a narrow window of their emission spectrum, in which many of these fluorochromes overlap, limiting the number of markers that can be measured. In contrast, spectral flow cytometry acquires the full emission spectrum of each fluorochromes, which enables high resolution characterization of fluorochromes (or fluorescent proteins) that can be used in conjunction. Moreover, the autofluorescence emitted by unstained cells can also be acquired and subtracted for better detection of weaker fluorescent signals. The 5-laser Aurora from Cytek is one of the most powerful spectral flow cytometry instruments currently available and the need for this technology is supported (1) by the fact that our two most high-end conventional flow cytometers (LSRII) are reaching end of serviceable life in one year, and (2) by increasing demand for greater capabilities, including more parameters analyzed per sample, as well as better sensitivity for dim markers and for small particles. With the 5-laser Aurora, investigators can expand the number of markers assessed to well beyond 30 markers: Cytek has validated a 40-color panel for human blood and Dr. Farber at the Columbia University Irving Medical Center (CUIMC) is applying panels of 25-35 parameters for several of her projects with several substitutions made possible by the flexibility of panel design. This capability is attractive to many Flow Cytometry Core users who have limited amount of precious material to work with, including cells isolated from small tissue biopsies, humanized mice (particularly blood and lymph node samples), patient airway washes and tissue organoids. This technology is also ideal as an upgrade option for most of our LSRII users, as opposed to mass cytometry, which requires acquiring or generating all new reagents. When testing the Aurora, users have been able to overcome major roadblocks in their projects, such as the ability to identify tissues cells whose autofluorescence was masking the expression of a fluorescent reporter. Users also validated the detection of particles or cell products in the 100-200 nm size range, made possible by the Aurora's violet side scatter detector. In this proposal, 34 principal investigators at CUIMC have indicated how their projects (84% of which are NIH- funded) will be significantly enhanced by upgrading from conventional flow cytometry instrument to the 5-laser Aurora. Thus, the addition of this instrument will have a beneficial and long-lasting impact on biomedical research encompassing a broad variety of fields. Generous institutional support is provided in the form of incentives and coverage of maintenance costs, and the operation of the 5-laser Aurora will be sustainable in the long-term because its maintenance is comparable to conventional instruments in use.

项目概要 流式细胞术是对多种细胞类型进行单细胞分析以评估 表面和细胞内标记的表达并进行功能测量。传统流式细胞术 一直无法利用多种可用于标记分析的荧光染料，因为 技术专注于其发射光谱的窄窗口，其中许多荧光染料 重叠，限制了可测量的标记数量。相比之下，光谱流式细胞术获得 每种荧光染料的完整发射光谱，可实现荧光染料的高分辨率表征 （或荧光蛋白）可以结合使用。此外，未染色的样品发出的自发荧光 还可以获取并减去细胞，以便更好地检测较弱的荧光信号。 Cytek 的 5 激光 Aurora 是目前最强大的光谱流式细胞仪之一 我们的两个最高端的传统流程支持了对这项技术的需求 (1) 细胞计数仪（LSRII）将在一年内达到使用寿命，并且（2）随着对更大容量的需求的增加 功能，包括每个样本分析更多参数，以及对暗淡标记和 对于小颗粒。借助 5 激光 Aurora，研究人员可以将评估的标记物数量扩大到 超过 30 个标记：Cytek 和哥伦比亚大学法伯博士验证了人类血液的 40 色面板 欧文大学医学中心 (CUIMC) 正在为她的几个项目应用 25-35 个参数的面板 面板设计的灵活性使得多种替代成为可能。这个能力对很多 Flow 来说很有吸引力 Cytometry Core 用户的珍贵材料有限，包括从细胞中分离出来的细胞 小组织活检、人源化小鼠（特别是血液和淋巴结样本）、患者气道冲洗和 组织类器官。对于我们大多数 LSRII 用户来说，这项技术也是理想的升级选项，而不是 质谱流式细胞术，需要获取或生成所有新试剂。在测试 Aurora 时，用户有 能够克服项目中的主要障碍，例如识别组织细胞的能力 自发荧光掩盖了荧光报告基因的表达。用户还验证了检测 100-200 nm 尺寸范围内的颗粒或细胞产物，通过 Aurora 的紫色侧散射探测器成为可能。 在这份提案中，CUIMC 的 34 位主要研究人员表明了他们的项目（其中 84% 是 NIH- 通过从传统的流式细胞仪升级到 5 激光，将显着增强 极光。因此，该仪器的加入将对生物医学研究产生有益且持久的影响 涵盖广泛的各个领域。以激励和激励的形式提供慷慨的机构支持 覆盖维护成本，并且 5 激光器 Aurora 的运行将长期可持续 因为它的维护与使用中的传统仪器相当。