MRI: Acquisition of FACSARIA-II, a next-generation high-speed cell sorter for a Flow Cytometry Core supporting interdisciplinary research and training

流式细胞术是一种强大的分析技术，有助于基于固有或操纵细胞属性的细胞群体的表征，定量和/或分离。用荧光偶联抗体或荧光活性染料在外部或内部标记细胞，或通过基因表达荧光报告分子，都是定义细胞群的策略，可以使用流式细胞仪进行分化。近年来，BectonDickinson取得了重大的技术进步，凭借其FACSAria(4)平台，在创建下一代细胞分选器方面处于领先地位。FACSAriaII是一种新的、用户友好的台式高速细胞分选仪，具有数字电子器件，可基于多达14个参数（包括细胞大小和内部复杂性）进行高速分析（70,000事件/秒），同时对两个或四个种群进行高速分选，为用户节省了时间和资源。样品喷嘴很容易互换，以适应分选大细胞。它的固定激光对准提高了灵敏度，最大限度地减少了启动时间，提高了实验之间的可重复性，并实现了自动化的日常质量控制。它能够无菌分选细胞，用于后续的细胞培养。FACSAriaII平台是可升级的，允许一开始就进行经济配置，并保留灵活性以满足未来不断变化的用户需求。高速分选，大细胞尺寸调节，提高灵敏度和可升级性，使FACSAriaII现在的最佳选择和一个很好的长期投资。该提案描述了作为facsarii主要用户的9个pi和另外10个次要用户的研究。他们的项目，根据他们对facsariai固有的一种或多种独特功能的需求进行了广泛的分组：1)独特目标细胞群的高速分选；Ii)同时，高速富集多个细胞群，以进一步在体内或离体利用；Iii)需要多参数分层的高速分选。许多这些pi都优化了协议，确保在获得facsariai后几乎立即获得数据。试剂开发是许多pi的另一个重要目标，获得facsariai将为他们提供推进和加速整个研究领域发现的能力。化学工程师将在每个细胞的基础上修改和分析植物细胞，以改善它们治疗癌症的化合物的生产，或者为合成生物学和工业生物技术设计细菌群体感应。生物学家将能够更好地了解调节植物生长结构动力学的基因，化学家将使用细菌表达系统来生成和筛选合成的变构开关。可以调节细胞死亡。聚合物科学家将跟踪合成有效载荷进入活细胞的效率和后果，而免疫学家将恢复和操纵稀有细胞群，将它们从一个宿主转移到另一个宿主，以更好地定义疾病机制，确定新的治疗靶点，或推进疫苗开发。FACSAriaII高速细胞分选器的能力将加强和加速正在进行的、多样化的、在许多情况下是跨学科的研究。许多拟议的研究项目都有应用目标，包括潜在的转化效益，例如确定新的治疗靶点和为新的有效载荷递送提供原理验证数据。此外，其中一些项目可能具有工业或商业应用。现场访问facssari将加速实现这些目标的进展。主要和次要用户群体共同培养了许多本科生和研究生，其中许多人通过资助的跨学科教育和培训，如马萨诸塞大学/阿默斯特细胞工程研究所和化学/生物接口。展望未来，通过与五所学院联盟中的姐妹学院的联系，Flow Core设施将为培养流式细胞术技术的本科生提供更多机会，包括来自Mt. Holyoke和Smith学院的学生，这两所女子学院的本科生中有很大一部分是为STEM领域的高级培训做准备的。具体来说，与史密斯学院的暑期科学与工程项目（每年向全国各地的高中年龄的年轻女性提供）相结合，将开发一个流式细胞术和细胞分选研究模块，向下一代女科学家介绍单细胞分析的力量。回顾过去，马萨诸塞大学/阿默斯特分校长期以来在招募和留住代表性不足的群体方面享有盛誉，部分原因是它在东北研究生教育和教授联盟中发挥了主导作用。这个由五个机构组成的组织致力于为代表性不足的群体扩大科学研究生教育和职业发展的机会。2008年，卡内基基金会认可了马萨诸塞大学/阿默斯特分校的外展努力，并将马萨诸塞大学指定为社区参与大学。FACSAriaII是一种将强大的单细胞分选和分析与易于使用的用户友好设计相结合的仪器，将在继续这些努力中发挥宝贵的作用。

Flow cytometry is a powerful analytic technique that facilitates the characterization, quantification and/or isolation of cell populations based on innate or manipulated cellular attributes. Labeling cells externally or internally with fluorescently-conjugated antibodies or fluorescent vital dyes, or by genetically expressing fluorescent reporter molecules, all represent strategies for defining cell populations that may be differentiated using a flow cytometer.In recent years, BectonDickinson has made significant technological improvements, leading the field in creating the next-generation of cell sorters with its FACSAria (4) platform. A newer, user-friendly, bench-top high-speed cell sorter, the FACSAriaII, has digital electronics that allows high-speed analyses (70,000 events/second) based on up to 14 parameters, including cell size and internal complexity, with concomitant high-speed, simultaneous sorting of two or four populations, saving time and resources for users. Sample nozzles are easily interchanged to accommodate sorting large cells. Its fixed-laser alignment increases sensitivity, minimizes start-up time, improves reproducibility between experiments and enables automated daily quality control. It is capable of sorting cells aseptically for subsequent cell culture. The FACSAriaII platform is upgradeable, permitting economic configuration at the outset and retaining the flexibility to meet evolving user needs in the future. High-speed sorting, large cell size accommodation, increased sensitivity and upgradability make the FACSAriaII the best possible choice now and an excellent long-term investment.This proposal describes the research of nine PIs as major users of a FACSAriaII, and an additional 10 minor users. Their projects, are broadly grouped by their requirements for one or more of the unique capabilities intrinsic to the FACSAriaII: i) high-speed sorting of unique target cell populations; ii) simultaneous, high-speed enrichment of multiple cell populations for further in vivo or ex vivo utilization; iii) high-speed sorting requiring multi-parameter stratification. Many of these PIs have optimized protocols in place, ensuring data will be obtained almost immediately upon acquiring a FACSAriaII. Reagent development is another important goal of many of the PIs, and acquiring a FACSAriaII will provide them with the capacity to advance and accelerate discovery for entire fields of research.Chemical engineers will modify and analyze plant cells, on a per cell basis, to improve their production of cancer-treating compounds, or engineer bacterial quorum sensing for synthetic biology and industrial biotechnology. Biologists will be able to better understand the genes that regulate the structural dynamics of plant growth and chemists will use bacterial expression systems to generate and screen for synthetic allosteric ?switches? that can regulate cell death. Polymer scientists will track the efficiency and consequences of synthetic payload uptake into viable cells, while immunologists will recover and manipulate rare cell populations, transferring them from one host to another to better define mechanisms of disease, identify novel therapeutic targets, or advance vaccine development. Ongoing, diverse and, in many cases, interdisciplinary research will be strengthened and accelerated by the capabilities of a FACSAriaII high-speed cell sorter. Many of the proposed research projects have applied objectives including potential translational benefits, such as identifying novel therapeutic targets and providing proof-of-principle data for novel delivery of payload. Additionally, some of these projects may have industrial or commercial applications. On-site access to a FACSAriaII will hasten progress toward these objectives. The group of major and minor users collectively trains many undergraduate and graduate students, and many of these through grant-funded interdisciplinary education and training, such as the University of Massachusetts/ Amherst Institute for Cellular Engineering and the Chemistry/Biology Interface. Looking forward, through outreach to sister colleges in the Five College Consortium, the Flow Core Facility will provide expanded opportunities for training undergraduate students in flow cytometric techniques, including students from Mt. Holyoke and Smith College, two all-women colleges that prepare a significant percentage of their undergraduate enrollment for advanced training in the STEM fields. Specifically, in conjunction with Smith College's Summer Science and Engineering Program, offered each year to high school-aged young women from across the country, a flow cytometry and cell sorting research module will be developed to introduce the next generation of women scientists to the power of single-cell analysis. Looking back, the University of Massachusetts/Amherst has a long-standing reputation for recruiting and retaining under-represented groups, in part through its lead role in the Northeast Alliance for Graduate Education and the Professoriate. This five-institution organization is committed to expanding opportunities for scientific graduate education and career advancement for under-represented groups. In 2008, the Cargnegie Foundation recognized the outreach efforts of the University of Massachusetts/Amherst when it designated UMass a Community Engaged University. The FACSAriaII, an instrument that combines powerful single-cell sorting and analysis with an accessible, user-friendly design will play an invaluable role in continuing these efforts.