Application of analytical cytology to current issues in the biomedical sciences

流式细胞仪共享资源设施(FCU)倡议：由于流式细胞仪的核心责任，这些项目被搁置。 虽然流式细胞仪核心的用户在这项技术方面拥有广泛的专业知识，但我们经常遇到他们的试剂板的设计和实施不是最优的情况。我们意识到，一篇记录选择补偿试剂和进行适当的流式细胞仪补偿以进行样本验证的适当策略的论文将使我们在IRP的用户基础以及整个流式细胞仪社区受益。我们对典型的补偿试剂进行了全面的分析，即在抗体面板中使用相同试剂的单色抗体对照，并将其扩展到使用与面板中的每个荧光染料结合的CD8和CD45抗体，并与Spherotech Inc.合作，使用抗原涂层珠子作为补偿对照。这项以人体为中心的研究还利用了大量的抗体面板(12)，从简单的(4色)到6色和8色，包括违反抗体荧光选择的常见流式细胞术规则的面板，商业面板，以及用于NIH反式免疫学模拟的Euroflow面板。数据采集完成，对结果的初步审查表明，在许多情况下，使用相同颜色的抗体(即小组中的CD28，使用CD28作为补偿对照)和CD45或CD8匹配的荧光色素对照是不合适的。结果还显示，这些珠子在制造商之间存在重大差异，增加了选择和设置补偿控制的错误。

Flow Cytometry Shared Resource Facility (FCU) initiatives: Because of flow cytometry core responsibilities, these projects are on hold. While users of the flow cytometry core have a broad range of expertise in this technology, we frequently have encountered situations where the design and implementation of their reagent panels is less than optimal. We realized that a paper documenting the proper strategies for selecting both compensation reagents and performing proper flow cytometric compensation for sample validation would benefit both our user base in the IRP as well as the flow cytometry community at large. We undertook a comprehensive analysis of typical compensation reagents, i.e. single color antibody controls using the same reagents in an antibody panels and expanded this to the use of CD8 and CD45 antibodies conjugated to each of the fluorochromes in the panel, and in collaboration with Spherotech Inc., the use of antigen-coated beads as compensation controls. This human-focused study also utilized a large number of antibody panels (12) that ranged from the simple (4-color), to 6- and 8-color, including panels that violated the common flow cytometric rules of antibody-fluorochrome selection, commercial panels, and the Euroflow panels used in the NIH trans-immunology imitative. Data acquisition is completed and an initial review of the results indicates there are a number of instances where the use of the same color antibodies (i.e. CD28 in the panel with use of CD28 as a compensation control) and either CD45 or CD8-matched fluorochrome controls are inappropriate. The results also show major differences in these beads among manufacturers adding to errors in selecting and setting compensation controls.