Flow Cytometry Protocols

David W. Galbraith, Elwira Sliwinska, Partha Samadder

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.