Five-laser Aurora spectral flow cytometer

项目摘要 流式细胞术是用于多种细胞类型的单细胞分析的基本工具，以评估细胞周期的变化。 表面和细胞内标记物的表达并进行功能测量。常规流式细胞术 一直无法利用可用于标记物分析的大量荧光染料，因为 技术集中在它们发射光谱的窄窗口上，其中许多荧光染料 重叠，限制了可以测量的标记的数量。相比之下，光谱流式细胞术获得了 每种荧光染料的全发射光谱，可实现荧光染料的高分辨率表征 (or荧光蛋白），其可以结合使用。此外，未染色的细胞发出的自发荧光 也可以获取和减去细胞，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 对这项技术的需求得到了以下事实的支持：（1）我们的两个最高端的传统流程 血细胞计数器（LSRII）在一年内就将达到使用寿命，（2）由于对更大容量血细胞计数器的需求增加， 功能，包括每个样品分析的更多参数，以及对暗淡标记的更高灵敏度， 对于小颗粒。使用5激光Aurora，研究人员可以将评估的标记物数量扩展到 超过30个标记：Cytek已经验证了一个40色的人类血液和法伯博士在哥伦比亚 大学欧文医学中心（CUIMC）正在为她的几个项目应用25-35个参数的面板， 由于面板设计的灵活性，可以进行几种替换。这种能力对许多Flow具有吸引力 细胞计数核心用户的珍贵材料数量有限，包括从 小组织活组织检查、人源化小鼠（特别是血液和淋巴结样品）、患者气道洗涤和 组织类器官对于大多数LSRII用户来说，该技术也是理想的升级选项， 质谱流式细胞术，这需要获取或生成所有新试剂。在测试Aurora时， 能够克服他们项目中的主要障碍，例如识别组织细胞的能力， 自体荧光掩盖了荧光报告基因的表达。用户还验证了 颗粒或细胞产物在100-200纳米的大小范围内，使极光的紫色侧散射检测器成为可能。 在这份提案中，CUIMC的34名主要研究者指出了他们的项目（其中84%是NIH- 通过从传统流式细胞仪升级到5-激光， 奥罗拉因此，这一工具的加入将对生物医学研究产生有益和持久的影响 涵盖广泛的领域。慷慨的机构支持以奖励的形式提供， 维护成本的覆盖范围，5激光极光的运营将是长期可持续的 因为它的维护与使用中的传统仪器相当。

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.