Analysis of DNA Content and BrdU Incorporation

Zbigniew Darzynkiewicz1, Gloria Juan1

1 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.7
DOI:  10.1002/0471142956.cy0707s02
Online Posting Date:  May, 2001
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Abstract

During the past two decades, the techniques of analysis of cell cycle kinetics based upon the flow cytometric detection of BrdU incorporation have replaced the cumbersome autoradiographic methodology utilizing tritiated thymidine. This unit distills the knowledge gained over this time into concise and easy to follow protocols for DNA content analysis following thermal denaturation or acid denaturation, as well as a modified method that combines thermal denaturation with washless immunocytochemical detection of BrdU.

     
 
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Table of Contents

  • Basic Protocol 1: Analysis of DNA Content and BrdU Incorporation Following Thermal Denaturation of DNA
  • Basic Protocol 2: Analysis of DNA Content and BrdU Incorporation Following Acid Denaturation of DNA
  • Alternate Protocol 1: Indirect Immunocytochemical Method for BrdU Detection
  • Alternate Protocol 2: “Washless” Technique for BrdU Detection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Analysis of DNA Content and BrdU Incorporation Following Thermal Denaturation of DNA

  Materials
  • 1 mg/ml BrdU stock solution in distilled water (prepare fresh)
  • Cells to be labeled and appropriate serum‐containing tissue culture medium ( appendix 3B)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% ethanol, ice‐cold
  • 0.1% Triton X‐100/0.1 M HCl, ice‐cold (prepare fresh)
  • DNA denaturation buffer (see recipe)
  • Antibody diluting buffer (see recipe)
  • 0.2 to 0.5 µg/100 µl FITC‐conjugated anti‐BrdU antibody (Becton Dickinson Immunocytometry) in antibody diluting buffer (see recipe for buffer)
  • Propidium iodide (PI) staining solution (see recipe)
  • Tabletop centrifuge
  • 15‐ml tubes (preferably glass)
  • 85° to 95°C water bath
  • 25‐G needle
  • Flow cytometer with 488‐nm laser line filter or BC 12 blue filter as well as 530 ± 20–nm bandpass filter and 620‐nm long‐pass filter
  • Additional reagents and equipment for trypsinizing tissue culture cells ( appendix 3B)

Basic Protocol 2: Analysis of DNA Content and BrdU Incorporation Following Acid Denaturation of DNA

  Materials
  • 1 mg/ml BrdU stock solution in distilled water (prepare fresh)
  • Cells to be labeled and appropriate serum‐containing tissue culture medium ( appendix 3B)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 70% ethanol, ice‐cold
  • 2 M HCl
  • Phosphate/citric acid buffer, pH 7.4 (see recipe)
  • 0.2 to 0.5 µg/100 µl FITC‐conjugated anti‐BrdU antibody (Becton Dickinson Immunocytometry) in antibody diluting buffer (see recipe for buffer)
  • Antibody diluting buffer (see recipe)
  • Propidium iodide (PI) staining solution (see recipe)
  • Flow cytometer with 488‐nm laser line filter or BC 12 blue filter as well as 530 ± 20–nm bandpass filter and 620‐nm long‐pass filter

Alternate Protocol 1: Indirect Immunocytochemical Method for BrdU Detection

  • 0.2 to 0.5 µg/100 µl mouse anti‐BrdU antibody (unlabeled) in antibody diluting buffer (see recipe for buffer)
  • 0.5 to 1 µg/ml FITC‐conjugated goat anti‐mouse antibody in recipeantibody diluting buffer
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Figures

Videos

Literature Cited

Literature Cited
   Begg, A.C., McNally, N.J., Shrieve, D.C., and Karcher, H. 1985. A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6:620‐626.
   Beisker, W., Dolbeare, F., and Gray, J.W. 1987. An improved immunocytochemical procedure for high sensitivity detection of incorporated bromodeoxyuridine. Cytometry 8:235‐239.
   Crissman, H.A. and Steinkamp, J.A. 1994. Cytochemical techniques for multivariate analysis of DNA and other cellular constituents. In Flow Cytometry and Sorting (M.R. Melamed, T. Lindmoand M.L. Mendelsohn, eds.) pp. 227‐247. Wiley‐Liss, New York.
   Darzynkiewicz, Z. 1990. Probing nuclear chromatin by flow tometry. In Flow Cytometry and Sorting M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 315‐340. Wiley‐Liss, New York.
   Darzynkiewicz, Z., Andreeff, M., Traganos, F., and Melamed, M.R. 1978. Discrimination of cycling and noncycling lymphocytes by BudR‐suppressed acridine orange fluorescence in a flow cytometric system. Exp. Cell Res 115:31‐ 35.
   Darzynkiewicz, Z., Robinson, J.P., and Crissman, J.P. (eds.) 1994. Flow Cytometry, 2nd ed part A. Academic Press, San Diego.
   Dolbeare, F. and Gray, J.W. 1988. Use of restriction endonuclease and exonuclease III to expose halogenated pyrimidines to immunocytochemical staining. Cytometry 9:631‐ 635.
   Dolbeare, F. and Selden, J.R. 1994. Immunocytochemical quantitation of bromodeoxyuridine: Application to cell‐cycle kinetics. Methods Cell Biol. 41:298‐ 316.
   Dolbeare, F., Gratzner, H., Pallavicini, M., and Gray, J.W. 1983. Flow cytometric measurement of total DNA content and incorporated bromodeoxyuridine. Proc. Natl. Acad. Sci. U.S.A 80:5573‐5577.
   Gratzner, H.G. 1982. Monoclonal antibody to 5‐bromo‐2‐deoxyuridine: A new reagent for detection of DNA replication. Science 218:747‐748.
   Gray, J.W., Dolbeare, F., and Pallavicini, M.G. 1990. Quantitative cell cycle analysis. In Flow Cytometry and Sorting (M.R. Melamed, T. Lindmo, and M.L. Mendelsohn, eds.) pp. 445‐467. Wiley‐Liss, New York.
   Larsen, J.R. 1994. Washless procedures for nuclear antigen detection. Methods Cell Biol 41:377‐388.
   Larsen, J.K., Christensen, I.J., Christiansen, J., Mortensen, T. 1991. Washless double staining of unfixed nuclei for flow cytometric analysis of DNA and a nuclear antigen (Ki‐67 or bromodeoxyuridine). Cytometry 12:429‐437.
   Latt, S.A. 1973. Microfluorimetric detection of deoxyribonucleic acid replication in human metaphase chromosomes. Proc. Natl. Acad. Sci. U.S.A. 70:3395‐3399.
   Latt, S.A. 1977. Fluorometric detection of deoxyribonucleic acid synthesis: Possibilities for interfacing bromodeoxyuridine dye techniques with flow cytometry. J. Histochem. Cytochem 25:913‐926.
   Li, X. and Darzynkiewicz, Z. 1995. Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28:571‐579.
   Li, X., Melamed, M.R., and Darzynkiewicz, Z. 1996. Detection of apoptosis and DNA replication by differential labeling of DNA strand breaks with fluorochromes of different color. Exp. Cell Res. 222:28‐37.
   Moran, R., Darzynkiewicz, Z., Staiano‐Coico, L., and Melamed, M.R. 1985. Detection of 5‐bromodeoxyuridine (BrdUrd) incorporation by monoclonal antibodies: Role of DNA denaturation step. J. Histochem. Cytochem. 33:821‐827.
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