Analysis of DNA Content and DNA Strand Breaks for Detection of Apoptotic Cells

Zbiginew Darzynkiewicz1, Gloria Juan1

1 New York Medical College, Valhalla, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.4
DOI:  10.1002/0471142956.cy0704s00
Online Posting Date:  May, 2001
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Abstract

This unit describes a method for identifying apoptotic cells based on in situ detection of DNA strand breaks. Correlated analysis of DNA content and DNA strand breaks allows one not only to identify apoptotic cells but also to pinpoint their location in the cell cycle. The basic protocol uses 5‐bromodeoxyuridine triphosphate to label the 3‐OH termini of the breaks, but the procedure is easily adapted to a variety of other conjugates. Given the multiplicity of commercially available reagents it is possible to label DNA strand breaks with a dye of any fluorescence color and excitation wavelength.

     
 
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Table of Contents

  • Basic Protocol 1: DNA Strand Break Labeling with BrdUTP
  • Alternate Protocol 1: Other Fluorochromes and Strategies for Labeling DNA Strand Breaks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: DNA Strand Break Labeling with BrdUTP

  Materials
  • Cells to be analyzed
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • 1% (w/v) methanol‐free formaldehyde (Polysciences) in PBS, pH 7.4
  • 70% ethanol, ice cold
  • TdT reaction buffer (see recipe)
  • BrdUTP stock solution: 2 mM BrdUTP (Sigma; 100 nmol in 50 µl) in 50 mM Tris⋅Cl ( appendix 2A), pH 7.5, made freshly and protected from light
  • 25 U/µl TdT in storage buffer (Boehringer Mannheim)
  • 10 mM CoCl 2 (Boehringer Mannheim)
  • Rinsing buffer: 0.1% (v/v) Triton X‐100 and 5 mg/ml BSA in PBS, pH 7.4 (can be stored at 4°C)
  • FITC‐conjugated anti‐BrdU MAb solution (see recipe)
  • PI staining buffer: 5 µg/ml propidium iodide (PI) and 200 µg/ml DNase‐free RNase A ( appendix 2A) in PBS, pH 7.4, made freshly
  • Silanized or polypropylene 15‐ml conical tube
  • Flow cytometer equipped with 488‐nm argon laser or mercury arc lamp, with blue (BG 12) excitation filter (∼50% cut off at 470 nm)
  • Additional reagents and equipment for trypsinizing cells ( appendix 3B) or dissociating cells from tissues (unit 5.2)
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Figures

Videos

Literature Cited

Literature Cited
   Arends, M.J., Morris, R.G., and Wyllie, A.H. 1990. Apoptosis: The role of endonuclease. Am. J. Pathol. 136:593‐608.
   Compton, M.M. 1992. A biochemical hallmark of apoptosis: Internucleosomal degradation of the genome. Cancer Metast. Rev. 11:105‐119.
   Darzynkiewicz, Z. 1993. Mammalian cell‐cycle analysis. In The Cell Cycle: A Practical Approach (P. Fantes and R. Brooks, eds.) pp. 45‐68. IRL Press, Oxford.
   Darzynkiewicz, Z., Juan, G., Li, X., Gorczyca, W., Murakami, T., and Traganos, F. 1997. Cytometry in cell necrobiology. Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1‐20.
   Dive, C., Gregory, C.D., Phipps, D.J., Evans, D.L., Milner, A.E., and Wyllie, A.H. 1992. Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry. Biochim. Biophys. Acta 1133:275‐285.
   Dolbeare, F. and Selden, J.R. 1994. Immunochemical quantitation of bromodeoxyuridine: Application to cell kinetics. Methods Cell Biol. 41:297‐316.
   Gold, R., Schmied, M., Giegerich, G., Breitschopf, H., Hartung, H.P., Toyka, K.V., and Lassman, H. 1994. Differentiation between cellular apoptosis and necrosis by the combined use of in situ tailing and nick translation techniques. Lab. Invest. 71:219‐225.
   Gorczyca, W., Bruno, S., Darzynkiewicz, R.J., Gong, J., and Darzynkiewicz, Z. 1992. DNA strand breaks occurring during apoptosis: Their early in situ detection by the terminal deoxynucleotidyl transferase and nick translation assays and prevention by serine protease inhibitors. Int. J. Oncol. 1:639‐648.
   Gorczyca, W., Gong, J., Ardelt, B., Traganos, F., and Darzynkiewicz, Z. 1993b. The cell cycle related differences in susceptibility of HL‐60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53:3186‐3192.
   Gorczyca, W., Bigman, K., Mittelman, A., Ahmed, T., Gong, J., Melamed, M.R., and Darzynkiewicz, Z. 1993a. Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia 7:659‐670..
   Gorman, A., McCarthy, J., Finucane, D., Reville, W., and Cotter, T. 1996. Morphological assessment of apoptosis. In Techniques in Apoptosis: A User's Guide (T.G. Cotter and S.J. Martin, eds.) pp. 3‐21. Portland Press, London.
   Hotz, M.A., Gong, J., Traganos, F., and Darzynkiewicz, Z. 1994. Flow cytometric detection of apoptosis. Comparison of the assays of in situ DNA degradation and chromatin changes. Cytometry 15:237‐244.
   Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Keehnen, R.M.J., Pals, S.T., and van Oers, M.H.J. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression of B cells undergoing apoptosis. Blood 84:1415‐1420.
   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.
   Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., and Riccardi, C. 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139:271‐280.
   Oberhammer, F., Wilson, J.M., Dive, C., Morris, I.D., Hickman, J.A., Wakeling, A.E., Walker, P.R., and Sikorska, M. 1993. Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12:3679‐3684.
   Tabor, S. Template‐independent DNA polymerases. 1995. In Short Protocols in Molecular Biology, 3rd ed. (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.23‐3.24. John Wiley & Sons, New York.
   Wyllie, A.H., Arends, M.J., Morris, R.G., Walker, S.W., and Evan, G. 1992. The apoptosis endonuclease and its regulation. Semin. Immunol. 4:389‐398.
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