Five-laser Aurora spectral flow cytometer

项目总结 流式细胞术是对多种细胞类型的单细胞进行分析的必要工具，以评估 细胞表面和细胞内标志物的表达，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分析的大量荧光染料，因为 技术的重点是它们发射光谱的一个狭窄窗口，在这个窗口中，许多这些荧光染料 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，这使得荧光染料的高分辨率表征成为可能 (或荧光蛋白)可以结合使用。此外，未染色的自体荧光 还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是目前可用的最强大的光谱流式细胞仪之一 对这项技术的需求(1)得到了以下事实的支持：我们两个最高端的传统流量 细胞仪(LSRII)将在一年内达到使用寿命结束，以及(2)通过增加对更多 功能，包括每个样本分析更多的参数，以及对暗淡标记和 对于小颗粒。使用5激光极光，调查人员可以将评估良好的标记物数量扩大到 超过30个标记：Cytek已经验证了一种40色的人体血液面板，哥伦比亚大学的Farber博士 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几个替代成为可能。这种能力对许多流量都很有吸引力 细胞仪核心用户，可使用的贵重材料数量有限，包括从 小组织活检、人源化小鼠(特别是血液和淋巴结样本)、患者呼吸道冲洗和 组织器官。这项技术也是我们大多数LSRII用户的理想升级选项，而不是 需要获取或产生所有新试剂的质量细胞术。在测试Aurora时，用户可以 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记者的表达。用户还验证了对 Aurora的紫罗兰侧散射探测器使100-200 nm大小的颗粒或细胞产品成为可能。 在这份提案中，CUIMC的34名首席调查人员指出了他们的项目(其中84%是NIH- 资金)将通过从传统的流式细胞仪升级到5-激光器而显著增强 奥罗拉。因此，该仪器的加入将对生物医学研究产生有益和持久的影响 包括各种各样的领域。慷慨的机构支持是以激励和 支付维护费用，从长远来看，5激光极光的运行将是可持续的 因为它的维护可以与使用中的常规仪器相媲美。

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.