Application of analytical cytology to current issues in the biomedical sciences

流式细胞仪共享资源设施(FCU)计划 单细胞分选与聚合酶链式反应 基于用户调查的大力支持，该实验室于2010年获得了专门为单细胞RT-和qRT-PCR设计的Advalytix(Ampliggrid)技术。从流式细胞术(分选)的角度来看，细胞被单独分选到经过特殊处理的玻片上(每个细胞都有微孔)，然后在为显微镜载玻片设计的台式单元上进行qRT-PCR处理。该核心一直在为一些用户零星地将单细胞沉积到PCR板中，这是对该技术的重大改进。该产品线被卖给了贝克曼·库尔特，后者于2012年停止生产。为了解决这个问题，我确定了可能的替代载玻片(从最初的每片50-75美元到0.50美元)，扩展了用于分类384个油井载片的矩阵，并为设计用于与该系统一起工作的定制PCR试剂寻找替代来源。核心人员(华莱士)一直在验证分选的准确性(以前从未发表过)，并记录了BD(SIC Aria分选器)的可识别问题，以及在侧流入射角度较窄的MoFlo和入射角度为0的iCyt上分选的优势。PCR臂一直是有问题的，特别是因为封闭液已经停产，配方仍然被认为是合适的；实验室的Nguyen已经探索了几种替代方案，我们与LMG博士后Peter Sykoa的合作已经扩展到用多个引物验证单细胞qPCR，目标是将其扩展到涉及人类细胞器遗传学的翻译项目。 流式细胞术中补偿策略的比较分析 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。 人类干细胞的条形码技术 为了分离明确定义的人类多能干细胞(HPSCs)群体，对细胞表面蛋白的详细了解将显著提高它们的特性和翻译潜力。通过化学蛋白质组学的方法，我们建立了包含496个N-连接的人胚胎糖蛋白(HESCs)和诱导的PSCs(HiPSCs)的细胞表面蛋白质组清单。在人类成纤维细胞和50种其他细胞类型的背景下，揭示了hPSCs感兴趣的100种表面蛋白。通过正交法验证的&gt；30阳性和阴性标记为合理选择多能性和谱系标记、用于细胞分离的表位和用于鉴定假定的HiPSC系的试剂提供了实验依据。化学蛋白质组定义的表面组和转录组预测的表面组之间的比较差异直接导致发现STF-31，一种已报道的GLUT-1抑制剂，对hPSCs具有毒性，并有效地选择性地从混合培养物中消除hPSCs。 博赫勒KR，巴塔查里亚S，Kropp EM，Chuppa S，Riordan DR，Bausch-Fluck D，Burbridge PW，Wu JC，Wersto RP，Chan GC，Rao S，Wollscheid B，Gundry RL。干细胞报告3：185-203,2014

Flow Cytometry Shared Resource Facility (FCU) initiatives Single Cell Sorting and PCR Based upon strong support from a user survey, the laboratory acquired the Advalytix (Ampligrid) technology specifically designed for RT- and qRT-PCR on single cells in 2010. From the flow cytometry (sorting) perspective, cells are sorted individually onto specially treated slides (which have microwells for each cell) and then processed for qRT-PCR on a benchtop unit designed for microscope slides. The core has been performing single-cell deposition into PCR plates sporadically for a number of users and this is a significant improvement of the technique. The product line was sold to Beckman Coulter who discontinued it in 2012. To remediate this problem, I identified potential replacements for the slides (from the original price of $50-75 each to $0.50), expanding the matrix for sorting onto 384 well slides, and finding replacement sources for the custom PCR reagents designed to work with the system. Core personnel (Wallace) has been validating the accuracy of sorting (something that has not been published before) and has documented identifiable problems with BD (sic Aria sorters) and the advantages of sorting on a MoFlo with narrower side stream angle of incidence and on the iCyt with 0 angle of incidence. The PCR arm has been problematic, particularly since the sealing solution is out of production and the formula still considered propriety; Nguyen in the laboratory has explored several alternatives and our collaboration with a LMG postdoc, Peter Sykoa, has expanded into validating single cell qPCR with multiple primers with the goal to expand it to a translational project involving human organelle genetics. Comparative analysis of compensation strategies in flow cytometry 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. Bar-coding of human stem cells Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures. Boheler KR, Bhattacharya S, Kropp EM, Chuppa S, Riordan DR, Bausch-Fluck D, Burridge PW, Wu JC, Wersto RP, Chan GC, Rao S, Wollscheid B, Gundry RL. Stem Cell Reports 3:185-203, 2014