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Multiparameter Cell Cycle Analysis

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Flow Cytometry Protocols

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

Abstract

Cell cycle-related cytometry and analysis is an essential experimental paradigm for the cell biology of yeast, mammalian, and drosophila cells. Methods have not changed much for many years. The most common is DNA content analysis, which has been well-published and reviewed. Next most common is analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation, detected by specific antibodies – also well-published and reviewed. A new measurement approach to S phase labeling utilizes 5′-ethynyl-2′-deoxyuridine (EdU) incorporation and a chemical reaction to label substituted DNA. The approach is new, but published work indicates that it is equivalent to BrdU incorporation. Finally, multiple antibody labeling to detect epitopes on cell cycle-regulated proteins is the most complex of the cytometric cell cycle assays, requiring knowledge of the chemistry of fixation, the biochemistry of antibody–antigen reactions, and spectral compensation. Because all of this knowledge is relatively well presented, methodologically, in many papers and reviews, this chapter presents a bare-bones Methods section for one mammalian cell type and an extended Notes section, focusing on aspects that are problematic or not well described in the literature.

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Acknowledgments

This work was supported by grants from NCI, R01CA73413 to JWJ and P30CA43703 to Stan Gerson, which supports the Cytometry and Imaging Microscopy Core facility in which the cytometry is performed. Additional thanks go to Vince Shankey (Beckman Coulter), Chuck Goolsby (Northwestern University), David Hedley (Ontario Cancer Institute), and Elena Holden (Compucyte) for advice and comments on this work for many years; Sue Chow (Ontario Cancer Institute) on fixation and staining, and Bruce Bagwell and Ben Hunsberger (Verity Software House) for many innovations in WinList that facilitate the analytical part of the work.

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Authors and Affiliations

  1. Cytometry and Imaging Microscopy Core, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA

    James W. Jacobberger, R. Michael Sramkoski & Tammy Stefan

Authors
  1. James W. Jacobberger
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  2. R. Michael Sramkoski
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  3. Tammy Stefan
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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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Jacobberger, J.W., Sramkoski, R.M., Stefan, T. (2011). Multiparameter Cell Cycle Analysis. 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_11

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_11

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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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