Galbraith David W, Sliwinska Elwira, Samadder Partha
School of Plant Sciences, Bio5 Institute, University of Arizona, 341 Keating Building, 1657 E. Helen Street, Tucson, AZ, 85721, USA.
Laboratory of Molecular Biology and Cytometry, Department of Plant Genetics, Physiology and Biotechnology, UTP University of Science and Technology, Kaliskiego Ave. 7, 85-789, Bydgoszcz, Poland.
Methods Mol Biol. 2018;1678:371-392. doi: 10.1007/978-1-4939-7346-0_16.
Eukaryotes are defined by cells that contain a nucleus and other membrane-bound organelles. Cytometric analysis in situ, utilizing imaging, provides a useful understanding of the structure and function of the various subcellular components, particularly when combined with methods that preserve the living state. In terms of information provided by the observation of eukaryotic nuclei, imaging has provided a wealth of information about cellular multiplication. When organisms are present in multicellular form (tissues and organs), this property does not generally confound imaging cytometry. Multicellular eukaryotic species present immediate problems when being considered for analysis using flow cytometry which requires suspensions of single particles. Although some eukaryotic cell types exist as natural single cell suspensions (cf. the erythropoietic system), for other tissues and organs, strategies are required to produce single particle suspensions. This chapter illustrates the application of flow cytometry combined with confocal microscopy to analyze complex organs, focusing on properties of the plant nucleus, and then goes on to describe how suspensions of nuclei can be prepared from tissues and organs, and used for flow cytometric analysis of cellular and transcriptional states. The application of these techniques to animal species is also discussed with the implication that this strategy is universally applicable for the characterization of nuclei within tissues that cannot readily be converted into suspensions of cells.
真核生物是由含有细胞核和其他膜结合细胞器的细胞所定义的。利用成像技术进行原位细胞分析，有助于深入了解各种亚细胞成分的结构和功能，特别是与保持活细胞状态的方法相结合时。就通过观察真核细胞核所提供的信息而言，成像技术已经提供了大量有关细胞增殖的信息。当生物体以多细胞形式（组织和器官）存在时，这种特性通常不会干扰成像细胞术。当考虑使用需要单个颗粒悬浮液的流式细胞术对多细胞真核生物物种进行分析时，会立即出现问题。尽管某些真核细胞类型以天然单细胞悬浮液的形式存在（如造血系统），但对于其他组织和器官，则需要采取策略来制备单个颗粒悬浮液。本章阐述了流式细胞术与共聚焦显微镜相结合在分析复杂器官中的应用，重点关注植物细胞核的特性，接着描述了如何从组织和器官中制备细胞核悬浮液，并将其用于细胞和转录状态的流式细胞术分析。还讨论了这些技术在动物物种中的应用，这意味着该策略普遍适用于那些难以转化为细胞悬浮液的组织中细胞核的表征。
