Galbraith David W
School of Plant Sciences, University of Arizona, Tuczon, AZ, USA.
Methods Mol Biol. 2014;1062:509-37. doi: 10.1007/978-1-62703-580-4_27.
Flow cytometry, and the accompanying technology of cell sorting, represents an established and valuable experimental platform for the analysis of cellular populations. Applications involving higher plants, which started to emerge around 30 years ago, are now widely employed both to provide unique information regarding fundamental questions in basic and applied bioscience and to advance agricultural productivity in practical ways. Further developments of this platform are being actively pursued, promising additional advances in our understanding of the interactions of cells within the complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types and second that their individual cells are generally larger than those of mammals. This chapter focuses on the use of flow cytometry and cell sorting with the model species Arabidopsis thaliana, in particular addressing (1) fluorescence in vivo labeling of specific cell types, (2) fluorescence-activated sorting of protoplasts and nuclei, and (3) transcriptome analyses using sorted protoplasts and nuclei.
流式细胞术以及与之相关的细胞分选技术,是用于分析细胞群体的成熟且有价值的实验平台。大约30年前开始出现的涉及高等植物的应用,如今已被广泛采用,既用于提供有关基础和应用生物科学中基本问题的独特信息,也用于以实际方式提高农业生产力。该平台的进一步发展正在积极推进,有望在我们对复杂组织和器官内细胞相互作用的理解上取得更多进展。高等植物在流式细胞术分析方面提出了独特的挑战,首先是因为它们的器官和组织几乎无一例外都是不同细胞类型的三维集合体,其次是它们的单个细胞通常比哺乳动物的细胞大。本章重点介绍利用模式植物拟南芥进行流式细胞术和细胞分选,特别涉及(1)特定细胞类型的体内荧光标记,(2)原生质体和细胞核的荧光激活分选,以及(3)使用分选的原生质体和细胞核进行转录组分析。