Malaghan Institute of Medical Research, Wellington, New Zealand.
Cytek Biosciences, Fremont, California.
Curr Protoc Cytom. 2020 Mar;92(1):e70. doi: 10.1002/cpcy.70.
Technological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large (20+ parameters) flow cytometry panels. However, as panel complexity and size increase, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and analyzing such high-dimensional data. A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorophore across all lasers, rather than identifying only the peak of emission. Fluorophores with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection. Here, we highlight the specific characteristics of spectral flow cytometry and aim to guide users through the process of building, designing, and optimizing high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly overlapping dyes, and a logical selection process for optimal marker-fluorophore assignment is provided. © 2020 by John Wiley & Sons, Inc.
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（20 多个参数）流式细胞术面板的发展。然而，随着面板复杂性和规模的增加，设计高质量面板、访问能够容纳大量参数的仪器以及分析这种高维数据的难度也随之增加。最近的一项进展是光谱流式细胞术，与传统的流式细胞术相比，它可以区分每个荧光染料在所有激光下的全发射光谱，而不仅仅是识别发射峰。因此，具有相似发射最大值但具有不同非峰特征的荧光染料可以在同一个流式细胞术面板中得到容纳，从而在面板设计和荧光染料检测方面具有更大的灵活性。在这里，我们强调了光谱流式细胞术的具体特点，并旨在通过使用全面的逐步协议，指导用户构建、设计和优化高维光谱流式细胞术面板。我们还特别考虑了使用高度重叠染料的情况，并提供了最佳标记-荧光染料分配的逻辑选择过程。 © 2020 年由 John Wiley & Sons, Inc. 出版
