Malaghan Institute of Medical Research, Wellington, New Zealand.
Maurice Wilkins Centre for Molecular Biodiscovery, Auckland, New Zealand.
Curr Protoc. 2021 Sep;1(9):e222. doi: 10.1002/cpz1.222.
Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.
荧光流式细胞术的技术进步和对免疫系统复杂性的不断深入理解，导致了大型流式细胞术面板的发展，在单细胞水平上达到了多达 43 种颜色。然而，随着面板尺寸和复杂性的增加，设计和优化适合下游高维数据分析的成功高质量面板所需的细节也越来越多。与传统流式细胞仪不同，全光谱流式细胞仪测量每个荧光染料在所有激光下的整个发射光谱。这使得发射峰非常相似但整体光谱指纹独特的荧光染料能够结合使用，从而能够相对轻松地设计更大的面板。虽然已经发表了全光谱流式细胞术面板设计最佳实践的方案，但从理论上设计的面板到面板优化所需的必要步骤之间仍然存在知识差距。在这里，我们旨在通过使用全面的逐步方案，指导用户优化用于免疫表型分析的高维全光谱流式细胞术面板的理论。当出现问题时，这些方案也可用于对面板进行故障排除。该方法的实际应用举例说明了一个 24 色面板，用于鉴定人类外周血中的常规 T 细胞亚群。©2021 玛拉干研究所，Cytek Biosciences。Wiley Periodicals LLC 出版的当前方案。基本方案 1：最佳光谱参考对照的制备和评估 支持方案 1：抗体滴定 支持方案 2：改变仪器设置 基本方案 2：完全染色样品的去混合评估 基本方案 3：标记分辨率评估 支持方案 3：管理异质自发荧光 基本方案 4：使用专家门控和降维算法评估数据质量。
