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

Zbiginew Darzynkiewicz¹, Gloria Juan¹

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

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Materials

Basic Protocol: 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 CoCl₂ (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

Figure 7.4.1

Measurement of DNA content combined with detection of apoptotic cells identified by the presence of DNA strand breaks, demonstrating the analysis of cell sensitivity to apoptosis in relation to the cell cycle phase. Apoptosis of HL-60 cells was induced by exposure to UV light (UV), treatment with the DNA topoisomerase I inhibitor camptothecin (CPT), irradiation ( RAD), or treatment with the DNA topoisomerase II inhibitor fostriecin (FST) as described (Gorczyca et al., 1993b; Darzynkiewicz et al., 1997). Populations of apoptotic cells are distinguished by the presence of DNA strand breaks. Note that UV light preferentially induces apoptosis of G₁ cells, CPT of S phase cells, and irradiation of G₂/M cells, whereas fostriecin is less selective, killing cells in all phases of the cycle. The left-hand panel shows untreated control cells, including their DNA content frequency histogram (inset). Separation of apoptotic and nonapoptotic cell populations by gating allows one to analyze the cell cyle distribution within each of these populations using software for deconvoluting DNA content frequency histograms.

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Figure 7.4.2

Detection of apoptosis during treatment of leukemia. Apoptotic cells (Ap) characterized by the presence of DNA strand breaks were detected in the peripheral blood of a patient with acute myelogenous leukemia (AML), prior to (A) and 2 days (B) and 4 days (C) after treatment with the DNA topoisomerase I inhibitor topotecan. Populations of apoptotic cells are very heterogeneous with respect to the number of DNA strand breaks. Cells with low FITC (green) fluorescence (L; within the trapezoid window) represent nonapoptotic cell populations. Note that most apoptotic cells have a DNA content equivalent to that of G₁ cells.

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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.