Hugh Green Cytometry Centre, Malaghan Institute of Medical Research, Wellington, New Zealand.
Malaghan Institute of Medical Research, Wellington, New Zealand.
Methods Mol Biol. 2024;2779:99-124. doi: 10.1007/978-1-0716-3738-8_6.
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 40 markers at the single-cell level. Full spectrum flow cytometry, which measures the full emission range of all the fluorophores present in the panel instead of only the emission peaks, is now routinely used in laboratories around the world, and the demand for this technology is rapidly increasing. With the ability to use larger and more complex staining panels, optimized protocols are vital for achieving the best panel design, panel optimization, and high-dimensional data analysis outcomes. In addition, a better understanding of how to fully characterize the autofluorescence of the sample, coupled with an intelligent panel design approach, allows improved marker resolution on highly autofluorescent tissues or cells. Here, we provide optimized step-by-step protocols for full spectrum flow cytometry, covering panel design and optimization, autofluorescence evaluation and strategy selection, and methods for performing longitudinal studies.
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 40 个标记物。全光谱流式细胞术现在在世界各地的实验室中得到了常规应用，它可以测量面板中所有荧光染料的全发射范围，而不仅仅是发射峰，对这种技术的需求正在迅速增加。随着使用更大和更复杂的染色面板的能力的提高，优化的方案对于实现最佳的面板设计、面板优化和高维数据分析结果至关重要。此外，更好地理解如何充分描述样本的自发荧光，加上智能的面板设计方法，可以提高对高度自发荧光组织或细胞的标记物分辨率。在这里，我们提供了全光谱流式细胞术的优化分步方案，涵盖了面板设计和优化、自发荧光评估和策略选择，以及进行纵向研究的方法。
