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，其有助于基于先天或操纵的细胞属性来表征、定量和/或分离细胞群。使用荧光结合抗体或荧光活体染料在细胞外部或内部标记细胞，或通过基因表达荧光报告分子，都是确定可使用流式细胞仪进行分化的细胞群的策略。近年来，BectonDickinson在技术上取得了重大进步，其FACSAria（4）平台在创建下一代细胞分选仪领域处于领先地位。FACSAriaII是一种更新的、用户友好的台式高速细胞分选机，它具有数字电子设备，可以根据多达14个参数进行高速分析（70，000个事件/秒），包括细胞大小和内部复杂性，同时高速分选两个或四个种群，为用户节省时间和资源。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，提高了实验之间的再现性，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随后的细胞培养。FACSAriaII平台是可扩展的，允许在开始时进行经济配置，并保持灵活性，以满足未来不断变化的用户需求。高速分选、大细胞尺寸适应性、更高的灵敏度和可重复性使FACSAriaII成为目前最佳选择和出色的长期投资。该提案描述了对9个主要使用FACSAriaII的PI和另外10个次要使用者的研究。他们的项目根据其对FACSAriaII固有的一种或多种独特能力的要求进行了广泛的分组：i）独特靶细胞群的高速分选; ii）多个细胞群的同时高速富集，以进一步体内或离体利用; iii）需要多参数分层的高速分选。其中许多PI都有优化的方案，确保在获得FACSAriaII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FACSAriaII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，以提高癌症治疗化合物的产量，或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控植物生长结构动力学的基因，化学家将利用细菌表达系统来产生和筛选合成的变构蛋白。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摄取到活细胞中的效率和后果，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高速细胞分选仪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，如确定新的治疗靶点，并为有效载荷的新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FACSAriaII将加快实现这些目标的进展。主要和次要用户群体共同培训许多本科生和研究生，其中许多是通过赠款资助的跨学科教育和培训，如马萨诸塞州大学/阿默斯特细胞工程和化学/生物学接口研究所。展望未来，通过与五所大学联盟中的姐妹学院的联系，Flow Core Facility将为流式细胞技术的本科生提供更多的培训机会，包括来自Mt.霍利奥克和史密斯学院，这两所全女子学院为STEM领域的高级培训准备了相当大比例的本科入学人数。具体而言，与史密斯学院的暑期科学和工程计划，每年提供给来自全国各地的高中年龄的年轻女性，流式细胞术和细胞分选研究模块将开发介绍下一代女科学家的单细胞分析的力量。回顾过去，马萨诸塞州/阿默斯特大学长期以来一直以招收和留住代表性不足的群体而闻名，部分原因是其在东北研究生教育联盟和教授协会中发挥了主导作用。这五个机构的组织致力于扩大科学研究生教育和职业发展的机会，为代表性不足的群体。2008年，卡耐基基金会认可了马萨诸塞州/阿默斯特大学的外联工作，并指定马萨诸塞大学为社区大学。FACSAriaII是一种将强大的单细胞分选和分析与易于访问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.