Detection of DNA Strand Breaks in Apoptotic Cells by Flow- and Image-Cytometry

  • Zbigniew Darzynkiewicz
  • Hong Zhao
Part of the Methods in Molecular Biology book series (MIMB, volume 682)


Extensive DNA fragmentation that generates a multitude of DNA double-strand breaks (DSBs) is a hallmark of apoptosis. A widely used approach to identify apoptotic cells relies on labeling DSBs in situ with fluorochromes. Flow or image cytometry is then used to detect and quantify apoptotic cells labeled this way. We developed several variants of the methodology that is based on the use of exogenous terminal deoxynucleotidyl transferase (TdT) to label 3′-OH ends of the DSBs with fluorochromes, defined as the TUNEL assay. This chapter describes the variant based on DSBs labeling using 5-Bromo-2′-deoxyuridine-5′-triphosphate (BrdUTP) as a TdT substrate and the incorporated BrdU is subsequently detected immunocytochemically with anti-BrdU antibody. We also describe modifications of the protocol that allow using other than BrdUTP deoxyribonucleotides to label DSBs. Concurrent differential staining of cellular DNA and multiparameter analysis of cells by flow- or image cytometry enables one to correlate the induction of apoptosis with the cell cycle phase. Examples of the detection of apoptotic cells in cultures of human leukemic cell lines treated with TNF-α and DNA topoisomerase I inhibitor topotecan are presented. The protocol can be applied to the cells growing in vitro, treated ex vivo with cytotoxic drugs as well as to clinical samples.

Key words

Apoptosis DNA damage Flow cytometry Laser scanning cytometry Cell cycle Immuno-fluorescence BrdU 



Supported by NCI grant RO1 28 704.


  1. 1.
    Nagata, S. (2000) Apoptotic DNA fragmentation. Exp. Cell Res., 256, 12–18.PubMedCrossRefGoogle Scholar
  2. 2.
    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.PubMedGoogle Scholar
  3. 3.
    Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M.A., Lassota, P., and Traganos, F. (1992) Features of apoptotic cells measured by flow cytometry. Cytometry, 13, 795–808.PubMedCrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    Gavrieli, Y. Sherman, Y., and Ben-Sasson, S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol., 119, 493–501.PubMedCrossRefGoogle Scholar
  6. 6.
    Gorczyca, W., Tuziak, T., Kram, A., Melamed, M.R., and Darzynkiewicz, Z. (1994) Detection of apoptosis in fine-needle aspiration biopsies by in situ end-labeling of fragmented DNA. Cytometry, 15, 169–175.PubMedCrossRefGoogle Scholar
  7. 7.
    Li, X., Traganos, F., Melamed, M.R., and Darzynkiewicz, Z. (1995) Single step procedure for DNA strand break labeling. Detection of apoptosis and DNA replication. Cytometry, 20, 172–180.PubMedCrossRefGoogle Scholar
  8. 8.
    Li, X., and Darzynkiewicz, Z. (1995) Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif., 28, 571–579.PubMedCrossRefGoogle Scholar
  9. 9.
    Gorczyca, W., Gong, J., Ardelt, B., Traganos, F., and Darzynkiewicz, Z. (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor drugs. Cancer Res., 53, 3186–3192.PubMedGoogle Scholar
  10. 10.
    Pozarowski, P., Holden, E., and Darzynkiewicz, Z. (2006) Laser scanning cytometry: Principles and applications. Methods Mol. Biol., 319, 165–192.PubMedCrossRefGoogle Scholar
  11. 11.
    Li, X., and Darzynkiewicz, Z. (1999). The Schrödinger’s cat quandary in cell biology: integration of live cell functional assays with measurements of fixed cells in analysis of apoptosis. Exp. Cell Res., 249, 4–412.Google Scholar
  12. 12.
    Li, X., Du, L., and Darzynkiewicz, Z. (2000) Caspases are activated during apoptosis independently of dissipation of mitochondrial electrochemical potential. Exp. Cell Res., 257, 290–297.PubMedCrossRefGoogle Scholar
  13. 13.
    Huang, X., Okafuji, M., Traganos, F., Luther, E., Holden, E., and Darzynkiewicz, Z. (2004) Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by DNA crosslinking agent cisplatin. Cytometry A, 58A, 99–110.CrossRefGoogle Scholar
  14. 14.
    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.PubMedCrossRefGoogle Scholar
  15. 15.
    Bedner, E., Li, X., Kunicki, J., and Darzynkiewicz, Z. (2000) Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry. Cytometry, 41, 83–88.PubMedCrossRefGoogle Scholar
  16. 16.
    Deptala, A., Bedner, E., Gorczyca, W., and Darzynkiewicz, Z. (1998) Simple assay of activation of nuclear factor kappa B (NF-κB) by laser scanning cytometry (LSC). Cytometry, 33, 376–382.PubMedCrossRefGoogle Scholar
  17. 17.
    Gong, J., Traganos, F., and Darzynkiewicz, Z. (1994) A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem., 218, 314–319.PubMedCrossRefGoogle Scholar
  18. 18.
    Dolbeare, F., and Selden, J.R. (1994) Immunochemical quantitation of bromodeoxyuridine: Application to cell cycle kinetics. Methods Cell Biol., 41, 297–316.PubMedCrossRefGoogle Scholar
  19. 19.
    Gorczyca, W., Bigman, K., Mittelman, A., Ahmed, T., Gong, J., Melamed, M.R., and Darzynkiewicz, Z. (1993) Induction of DNA strand breaks associated with apoptosis during treatment of leukemias. Leukemia, 7, 659–670.PubMedGoogle Scholar
  20. 20.
    Li, X., Gong, J., Feldman, E., Seiter, K., Traganos, F., and Darzynkiewicz, Z. (1994) Apoptotic cell death during treatment of leukemias. Leuk. Lymph., 13, 65–72.CrossRefGoogle Scholar
  21. 21.
    Oberhammer, F., Wilson, J.W., 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 50 kb fragments prior to or in the absence of internucleosomal degradation of DNA. EMBO J., 12, 3679–3684.PubMedGoogle Scholar
  22. 22.
    Li, X., and Darzynkiewicz, Z. (2000) Cleavage of poly(ADP-ribose) polymerase measured in situ in individual cells: relationship to DNA fragmentation and cell cycle position during apoptosis. Exp. Cell Res., 255, 125–132.PubMedCrossRefGoogle Scholar
  23. 23.
    Bedner, E., Smolewski, P., Amstad, P., and Darzynkiewicz, Z. (2000) Activation of caspases measured in situ by binding of ­fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp. Cell Res., 260, 308–313.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Zbigniew Darzynkiewicz
    • 1
  • Hong Zhao
    • 1
  1. 1.Department of PathologyNew York Medical College, Brander Cancer Research InstituteValhallaUSA

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