Skip to main content
SpringerLink
Log in

Nuclear Cytometry: Analysis of the Patterns of DNA Synthesis and Transcription Using Flow Cytometry, Confocal Microscopy, and RNA Sequencing

  • Protocol
  • First Online:
Flow Cytometry Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1678))

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Galbraith DW, Harkins KR, Maddox JM, Ayres NM, Sharma DP, Firoozabady E (1983) Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220(4601):1049–1051. doi:10.1126/science.220.4601.1049

    Article  CAS  Google Scholar 

  2. Galbraith DW, Lambert GM (2012) High-throughput monitoring of plant nuclear DNA contents via flow cytometry. Methods Mol Biol 918:311–325. doi:10.1007/978-1-61779-995-2_16

    Article  CAS  Google Scholar 

  3. Zhang C, Gong FC, Lambert GM, Galbraith DW (2005) Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods 1(1):7. doi:10.1186/1746-4811-1-7

    Article  CAS  Google Scholar 

  4. Samadder P, Weng N, Doetschman T, Heimark RL, Galbraith DW (2016) Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states. Cytometry A 89(5):430–442. doi:10.1002/cyto.a.22847

    Article  CAS  Google Scholar 

  5. Galbraith DW, Janda J, Lambert GM (2011) Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type. Methods Mol Biol 699:407–429. doi:10.1007/978-1-61737-950-5_20

    Article  CAS  Google Scholar 

  6. Nagl W (1976) DNA endoreduplication and polyteny understood as evolutionary strategies. Nature 261(5561):614–615

    Article  CAS  Google Scholar 

  7. Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M, Hofte H (1997) Cellular basis of hypocotyl growth in Arabidopsis thaliana. Plant Physiol 114(1):295–305

    Article  CAS  Google Scholar 

  8. Dilkes BP, Dante RA, Coelho C, Larkins BA (2002) Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control. Genetics 160(3):1163–1177

    Google Scholar 

  9. Sliwinska E (2009) Nuclear DNA replication and seed quality. Seed Sci Res 19:15–25

    Article  CAS  Google Scholar 

  10. Sliwinska E, Mathur J, Bewley JD (2012) Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication. J Exp Bot 63(11):4165–4178. doi:10.1093/jxb/ers099

    Article  CAS  Google Scholar 

  11. D’Amato F (1984) Role of polyploidy in reproductive organs and tissues. In: Johri BM (ed) Embryology of angiosperms. Springer, Berlin, pp 519–566

    Chapter  Google Scholar 

  12. Galbraith DW, Harkins KR, Knapp S (1991) Systemic Endopolyploidy in Arabidopsis thaliana. Plant Physiol 96(3):985–989

    Article  CAS  Google Scholar 

  13. Brown JK, Lambert GM, Ghanim M, Czosnek H, Galbraith DW (2005) Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry. Bull Entomol Res 95(4):309–312

    Article  CAS  Google Scholar 

  14. Galbraith DW (2009) Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry A 75(8):692–698. doi:10.1002/cyto.a.20760

    Article  Google Scholar 

  15. Grindberg RV, Yee-Greenbaum JL, McConnell MJ, Novotny M, O'Shaughnessy AL, Lambert GM, Arauzo-Bravo MJ, Lee J, Fishman M, Robbins GE, Lin X, Venepally P, Badger JH, Galbraith DW, Gage FH, Lasken RS (2013) RNA-sequencing from single nuclei. Proc Natl Acad Sci U S A 110(49):19802–19807. doi:10.1073/pnas.1319700110

    Article  CAS  Google Scholar 

  16. Zhang C, Barthelson RA, Lambert GM, Galbraith DW (2008) Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiol 147(1):30–40. doi:10.1104/pp.107.115246

    Article  CAS  Google Scholar 

  17. Barthelson RA, Lambert GM, Vanier C, Lynch RM, Galbraith DW (2007) Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics 8:340. doi:10.1186/1471-2164-8-340

    Article  Google Scholar 

  18. Jacob Y, Michaels SD (2008) Peering through the pore: the role of AtTPR in nuclear transport and development. Plant Signal Behav 3(1):62–64

    Article  Google Scholar 

  19. Haenni S, Ji Z, Hoque M, Rust N, Sharpe H, Eberhard R, Browne C, Hengartner MO, Mellor J, Tian B, Furger A (2012) Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3′-end-seq. Nucleic Acids Res 40(13):6304–6318. doi:10.1093/nar/gks282

    Article  CAS  Google Scholar 

  20. Deal RB, Henikoff S (2010) A simple method for gene expression and chromatin profiling of individual cell types within a tissue. Dev Cell 18(6):1030–1040. doi:10.1016/j.devcel.2010.05.013

    Article  CAS  Google Scholar 

  21. Deal RB, Henikoff S (2011) The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana. Nat Protoc 6(1):56–68. doi:10.1038/nprot.2010.175

    Article  CAS  Google Scholar 

  22. Paddock SW (1999) An introduction to confocal imaging. Methods Mol Biol 122:1–34

    CAS  Google Scholar 

  23. Sliwinska E, Mathur J, Bewley JD (2015) On the relationship between endoreduplication and collet hair initiation and tip growth, as determined using six Arabidopsis thaliana root-hair mutants. J Exp Bot 66(11):3285–3295. doi:10.1093/jxb/erv136

    Article  CAS  Google Scholar 

  24. Sliwinska E, Bassel GW, Bewley JD (2009) Germination of Arabidopsis thaliana seeds is not completed as a result of elongation of the radicle but of the adjacent transition zone and lower hypocotyl. J Exp Bot 60(12):3587–3594. doi:10.1093/jxb/erp203

    Article  CAS  Google Scholar 

  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  26. Silcock DJ, Francis D, Bryant JA, Hughies SG (1990) Changes in nuclear DNA content, cell and nuclear size, and frequency of cell division in the cotyledons of Brassica napus L. during embryogenesis. J Exp Bot 41:401–407

    Article  CAS  Google Scholar 

  27. Sugimoto-Shirasu K, Roberts K (2003) “Big it up”: endoreduplication and cell-size control in plants. Curr Opin Plant Biol 6(6):544–553

    Article  CAS  Google Scholar 

  28. Jovtchev G, Schubert V, Meister A, Barow M, Schubert I (2006) Nuclear DNA content and nuclear and cell volume are positively correlated in angiosperms. Cytogenet Genome Res 114(1):77–82. doi:10.1159/000091932

    Article  CAS  Google Scholar 

  29. Davidson D, Pertens E, Eastman M (1978) Nuclear and cell sizes in different regions of root meristems of Zea mays. Ann Bot 42:1429–1438

    Article  Google Scholar 

  30. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6(5):377–382. doi:10.1038/nmeth.1315

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Development of methods for flow analysis and sorting of mouse organ nuclei was done with support from the National Cancer Institute of the U.S. National Institutes of Health (awards number R21 CA164690, and P30 CA023074) and the Arizona Department of Health (ABRC ABC 13530).

Author information

Authors and Affiliations

  1. School of Plant Sciences, Bio5 Institute, University of Arizona, 341 Keating Building, 1657 E. Helen Street, Tucson, AZ, 85721, USA

    David W. Galbraith & Partha Samadder

  2. 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

    Elwira Sliwinska

Authors
  1. David W. Galbraith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elwira Sliwinska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Partha Samadder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David W. Galbraith .

Editor information

Editors and Affiliations

  1. Flow Cytometry Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Teresa S. Hawley

  2. Department of Anatomy and Regenerative Biology, School of Medicine and Health Sciences, George Washington University, Washington, District of Columbia, USA

    Robert G. Hawley

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Galbraith, D.W., Sliwinska, E., Samadder, P. (2018). Nuclear Cytometry: Analysis of the Patterns of DNA Synthesis and Transcription Using Flow Cytometry, Confocal Microscopy, and RNA Sequencing. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 1678. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7346-0_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7346-0_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7344-6

  • Online ISBN: 978-1-4939-7346-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation