LGC, Queens Road, Teddington, Middlesex, TW11 0LY, United Kingdom.
Cytometry A. 2018 Jul;93(9):905-915. doi: 10.1002/cyto.a.23248. Epub 2017 Sep 22.
Flow cytometry immunophenotyping is a sensitive technique allowing rapid characterization of single cells within heterogeneous populations, but it includes several subjective steps during sample analysis that impact the development of standardized methodology. Proposed strategies to overcome these limitations include fluorescent cell barcoding (FCB), which facilitates multiplexed sample evaluation with increased data reproducibility whilst reducing labeling variation, materials, and time. To date, the FCB assay has found utility for analyzing the phosphorylation status of intracellular targets but has not been intensively employed for cellular immunophenotypic analyses using cell surface markers. In this study we developed a modified FCB assay for multiplexed analysis of human mesenchymal stromal cells (hMSCs) to evaluate the quality of these cells during bioprocessing. A panel of fluorochrome-conjugated antibodies was used to target 15 ubiquitously expressed or stage-specific markers together with a fixable viability dye eFluor 506 acting as the cell barcoding agent. Critical technical considerations and validation steps were presented in the context of monitoring hMSC status, defined by generic, and specific surface markers for cell identity and quality. It was found that at discrete passages, inter-analyst expression patterns between hMSCs cultures were similar, but in contrast, diverse marker expression was evident between passages. A side-by-side analysis of barcoded and non-barcoded cells demonstrated the potential of this technique for the rapid phenotypic characterization of cells exposed to different bioprocessing conditions. Additionally, the method incorporates fewer subjective factors; including sample preparation and instrument day-to-day variations and is customizable across a diversity of cell types. © 2017 International Society for Advancement of Cytometry.
流式细胞术免疫表型分析是一种敏感的技术，能够快速描述异质群体中的单个细胞，但在样本分析过程中包含几个主观步骤，这会影响标准化方法的发展。克服这些限制的策略包括荧光细胞条形码（FCB），它通过增加数据可重复性同时减少标记变化、材料和时间来促进多路复用样品评估。迄今为止，FCB 测定法已被用于分析细胞内靶标的磷酸化状态，但尚未被广泛用于使用细胞表面标志物进行细胞免疫表型分析。在这项研究中，我们开发了一种改良的 FCB 测定法，用于多路复用分析人间充质基质细胞（hMSC），以评估这些细胞在生物处理过程中的质量。使用一组荧光染料标记的抗体针对 15 个普遍表达或阶段特异性标记物，以及固定活度染料 eFluor 506，用作细胞条形码剂。在监测 hMSC 状态的背景下提出了关键的技术考虑因素和验证步骤，该状态由细胞身份和质量的通用和特定表面标记物定义。结果发现，在离散的传代中，hMSC 培养物之间的分析师间表达模式相似，但相反，不同的标记物表达在传代之间明显。对条形码化和非条形码化细胞进行的并排分析证明了该技术用于快速表型鉴定暴露于不同生物处理条件的细胞的潜力。此外，该方法包含较少的主观因素；包括样品制备和仪器日常变化，并且可跨多种细胞类型定制。©2017 国际细胞分析促进协会。
