Flow Cytometry

流式细胞术核心（FCC）将提供三项主要服务，以支持四个领域的科学目标。 拟议项目;使用细胞分选进行群体纯化，使用细胞因子进行细胞因子鉴定和定量 基于珠的细胞因子阵列（Luminex系统）和使用该细胞因子阵列的多光谱成像流式细胞术（MIFC）。 ImageStream系统。这些服务中的第一项，细胞分选，将使用BD DiVa完成。 FACSVantage高速分选机或最近收购的i-Cyt Reflection高速分选机。这两 这些工具完全能够对项目1、2和3的目标中描述的感兴趣的人群进行分类。 将评价血清/血浆、组织匀浆和培养上清液的细胞因子谱。 完成了！使用基于Luminex珠的多路复用技术。将筛选多达32种分析物， 以及特定感兴趣的分析物的定制面板，以适应所有四个目标 项目FCC将为这些检测提供样品制备、数据采集和数据分析。 第三，使用ImageStream成像流式细胞仪，多光谱图像以及统计 强大的数据将可用于评估和确认蛋白质共定位，细胞因子的产生， 活化，以及提供关于来自肺的细胞群的额外形态学信息， 脾、肝和淋巴结。此外，如果需要，联邦通信委员会有可用的仪器， 支持多色流式细胞术，超出项目负责人自己仪器的能力。 FCC还将为项目负责人提供荧光染料选择方面的咨询服务， 样品制备、数据分析和数据解释。同样，FCC核心主任将协调 与组织学核心主任（核心B）合作，确保为所有人提供全面且免费的服务 四个项目，用于评估病毒清除和组织损伤过程中的免疫相互作用。 流式细胞术核心将提供工具，以a）分离特定的细胞亚群，以进一步研究其作用 在该过程中，B）鉴定和定量驱动应答的分泌的细胞因子/趋化因子的环境，c） 捕获被识别为关键成分的特定细胞类型的形态学和荧光图像。

The Flow Cytometry Core (FCC) will provide three primary services to support the scientific aims of the four proposed projects; population purification using cell sorting, cytokine identification and quantification using bead based cytokine arrays (Luminex system), and multispectral imaging flow cytometry (MIFC) using the ImageStream system. The first of these services, cell sorting, will be accomplished using a BD DiVa FACSVantage high speed sorter or the recently acquired i-Cyt Reflection high speed sorter. Both of these instruments are full equipped to sort the populations of interest described in the aims of Projects 1, 2, and 3. Evaluation of cytokine profiles of serum/plasma, tissue homogenates and culture supernatants will be accomplished! using the Luminex bead based multiplexing technology. Screening of up to 32 analytes will be available as well as customized panels of analytes of specific interest to accommodate the aims of all four projects. The FCC will provide sample preparation, data acquisition and data analysis for these assays. Thirdly, with the use of the ImageStream imaging flow cytometer, multispectral images as well as statistically robust data will be available to evaluate and confirm protein colocalizations, cytokine production, and cellular activation, as well as provide additional morphological information on cellular populations from the lung, spleen, liver and lymph node. In addition, the FCC has available instrumentation should the need arise to support polychromatic flow cytometry in excess of the capability of the Project leaders' own instrumentation. The FCC will also provide consultative services to the Project leaders in terms of fluorochrome selection, sample preparations, data analysis, and data interpretation. Likewise, the FCC Core Director will coordinate with the Director of the Histology Core (Core B) to insure comprehensive and complimentary services for all four Projects for the evaluation of immune interactions in the process of viral clearance and tissue injury. Flow Cytometry Core will provide the tools to a)isolate specific subsets of cells to further investigate their role in this process, b) identify and quantify the milieu of secreted cytokines/chemokines driving the response, c) capture morphological and fluorescent images of specific cell types identified as key components.