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Analysis of Cell Proliferation and Cell Survival by Continuous BrdU Labeling and Multivariate Flow Cytometry

Martin Poot1,  M. Rosato1,  Peter S. Rabinovitch1

1University of Washington, Seattle, Washington

Publication Name: 
Current Protocols in Cytometry
Unit Number: 
Unit 7.14
DOI: 
10.1002/0471142956.cy0714s15
Online Posting Date: 
May, 2001
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Abstract

Halogenated deoxyuridines such as BrdU incorporated into DNA in place of thymidine can be detected by virtue of their ability to quench the fluorescence of Hoechst dyes. This property is the basis for a simple and reliable method of enumerating cells which have incorporated BrdU. Counterstaining with another suitable DNA dye permits resolution of cells in G1, S, and G2 phases of three consecutive cell cycles. In addition to this basic technique, the authors present newer protocols for proliferative analysis of GFP-expressing or cell surface antigen expressing cells versus nonexpressing cells in culture as well as one to determine the absolute number of proliferating and nonproliferating cells by calibration for the volume analyzed. With this technique the proliferative survival of cells can be determined.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Analysis of Cell Proliferation by Continuous 5-BrdU Incorporation Followed by Hoechst 33258 and Ethidium Bromide Flow Cytometry
  • Alternate Protocol 1: Analysis of Cell Proliferation of GFP-Expressing Versus Nonexpressing Cells in a Single-cell Culture
  • Alternate Protocol 2: Analysis of Cell Proliferation of Antigen-Expressing Versus Nonexpressing Cells
  • Alternate Protocol 3: Analysis of Proliferative Survival
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Analysis of Cell Proliferation by Continuous 5-BrdU Incorporation Followed by Hoechst 33258 and Ethidium Bromide Flow Cytometry

 Materials
  • 10 mM 5-bromodeoxyuridine (BrdU) in distilled water
  • Cells in appropriate culture medium containing 10% FBS (appendix 2A)
  • Generic Hoechst staining buffer (see recipe)
  • 10% (v/v) Nonidet P-40 (or IGEPAL, Sigma)
  • 1 mg/ml ethidium bromide (Sigma) in distilled water
  • 15-ml screw-capped centrifuge tubes
  • Flow cytometer with either a mercury arc lamp or an argon laser (tuned to 360 nm) as excitation source; alternatively, two time-resolved argon lasers (tuned to 360- and 488-nm excitation wavelengths, respectively) can be used
  • 12 × 75–mm polypropylene flow cytometer sample tubes
  • Computer and appropriate software for data collection and processing

Alternate Protocol 1: Analysis of Cell Proliferation of GFP-Expressing Versus Nonexpressing Cells in a Single-cell Culture

 Additional Materials (also see Basic Protocol)
  • GFP-containing vector/virus cells
  • 2% (w/v) paraformaldehyde solution (see recipe)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1% (w/v) saponin (Sigma) in PBS
  • 1 mg/ml 7-aminoactinomycin D (7-AAD; Calbiochem) in DMSO

Alternate Protocol 2: Analysis of Cell Proliferation of Antigen-Expressing Versus Nonexpressing Cells

 Additional Materials (also see Basic Protocol)
  • FBS-PBS: 2% fetal bovine serum and 0.1 % NaN2 in PBS
  • Primary and secondary antibodies for desired antigens
  • 2% (w/v) paraformaldehyde (see recipe)
  • Phosphate buffered saline (PBS; appendix 2A)
  • 1% (w/v) saponin (Sigma) in PBS
  • 1 mg/ml 7-aminoactinomycin D (7-AAD; Calbiochem) in DMSO

Alternate Protocol 3: Analysis of Proliferative Survival

 Additional Materials (also see Basic Protocol)
  • Chicken erythrocyte nuclei (CEN; BioSure Controls)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 7.14.1
    Bivariate cytogram of cultured human cells stained with Hoechst 33258 dye (x-axis) and ethidium bromide (y-axis) with chicken erythrocyte standard added (see Alternate Protocol 3). The signal cluster corresponding to resting cells is labeled G0/G1, the cluster of first-cycle cells (S + G2/M) is labeled as such, as are the G1, S, and G2/M cells in the 2nd and 3rd cell cycles, and the cluster of chicken erythrocyte nuclei is labeled CEN.

    View Image

Literature Cited

Literature Cited
    Kubbies, M. and Rabinovitch, P.S. 1983. Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes. Cytometry 3:276-281.
    Latt, S.A. 1973. Microfluorometric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. U.S.A. 70:3395-3399.
    Loontiens, F.G., Regenfuss, P., Zechel, A., Dumortier, L., and Clegg, R.M. 1990. Binding characteristics of Hoechst 33258 with calf thymus DNA, poly[d(A-T)], and d(CCGGAATTCCGG): Multiple stoichiometries and determination of tight binding with a wide spectrum of site affinities. Biochemistry 29:9029-9039.
    Poot, M., Schuster, A., and Hoehn, H. 1991. Cytostatic synergism between bromodeoxyuridine, bleomycin, cisplatin and chlorambucil demonstrated by a sensitive cell kinetic assay. Biochem. Pharmacol. 41:1903-1909.
    Poot, M., Hoehn, H., Kubbies, M., Grossmann, A., Chen, Y., and Rabinovitch, P.S. 1994. Cell-cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33358-ethidium bromide bivariate flow cytometry. Methods Cell Biol. 41:327-340.
    Rabinovitch, P.S. 1983. Regulation of human fibroblast growth rate by both noncycling cell fraction transition probability is shown by growth in 5-bromodeoxyuridine followed by Hoechst 33258 flow cytometry. Proc. Natl. Acad. Sci. U.S.A. 80:2951-2955.
    Rabinovitch, P.S., Kubbies, M., Chen, Y.C., Schindler, D., and Hoehn, H. 1988. BrdU-Hoechst flow cytometry: A unique tool for quantitative cell cycle analysis. Exp. Cell Res. 174:309-318.
    Watkins, S. 1989. Immunohistochemistry. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.6.1-14.6.13. John Wiley & Sons, New York.
     
 
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