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Flow Cytometry Protocols pp 407–429Cite as

Multiparametric Analysis, Sorting, and Transcriptional Profiling of Plant Protoplasts and Nuclei According to Cell Type

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 699))

Abstract

Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.

This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

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Acknowledgments

Part of the development of the methods described in this chapter was done with support from the NSF Plant Genome program.

Author information

Authors and Affiliations

  1. Department of Plant Sciences, University of Arizona, Tucson, AZ, USA

    David W. Galbraith, Jaroslav Janda & Georgina M. Lambert

Authors
  1. David W. Galbraith
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  2. Jaroslav Janda
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  3. Georgina M. Lambert
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Editor information

Editors and Affiliations

  1. Medical Center, Flow Cytometry Core Facility, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Teresa S. Hawley

  2. Medical Center, Dept. Anatomy & Regenerative Biology, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Robert G. Hawley

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Galbraith, D.W., Janda, J., Lambert, G.M. (2011). Multiparametric Analysis, Sorting, and Transcriptional Profiling of Plant Protoplasts and Nuclei According to Cell Type. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_20

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_20

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-949-9

  • Online ISBN: 978-1-61737-950-5

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