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Analysis of apoptosis by propidium iodide staining and flow cytometry

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Abstract

Since its introduction, the propidium iodide (PI) flow cytometric assay has been widely used for the evaluation of apoptosis in different experimental models. It is based on the principle that apoptotic cells, among other typical features, are characterized by DNA fragmentation and, consequently, loss of nuclear DNA content. Use of a fluorochrome, such as PI, that is capable of binding and labeling DNA makes it possible to obtain a rapid (the protocol can be completed in about 2 h) and precise evaluation of cellular DNA content by flow cytometric analysis, and subsequent identification of hypodiploid cells. The original protocol enhanced the capacity for a rapid, quantitative measure of cell apoptosis. For this reason, since its publication, the PI assay has been widely used, as demonstrated by the large number of citations of the original paper and/or the continuous use of the method in many laboratories.

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Figure 1: Measure of apoptotic thymocytes.
Figure 2: The quick method can be safely used with mouse thymocytes.

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References

  1. Wyllie, A.H. Apoptosis: an overview. Br. Med. Bull. 53, 451–465 (1997).

    Article  CAS  Google Scholar 

  2. Arends, M.J., Morris, R.G. & Wyllie, A.H. Apoptosis. The role of the endonuclease. Am. J. Pathol. 136, 593–608 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Sellins, K.S. & Cohen, J.J. Gene induction by gamma-irradiation leads to DNA fragmentation in lymphocytes. J. Immunol. 139, 3199–3206 (1987).

    CAS  PubMed  Google Scholar 

  4. Gong, J., Traganos, F. & Darzynkiewicz, Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal. Biochem. 218, 314–319 (1994).

    Article  CAS  Google Scholar 

  5. Nunez, R. DNA measurement and cell cycle analysis by flow cytometry. Curr. Issues Mol. Biol. 3, 67–70 (2001).

    CAS  PubMed  Google Scholar 

  6. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F. & Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279 (1991).

    Article  CAS  Google Scholar 

  7. Ormerod, M.G. Investigating the relationship between the cell cycle and apoptosis using flow cytometry. J. Immunol. Methods 265, 73–80 (2002).

    Article  CAS  Google Scholar 

  8. Darzynkiewicz, Z., Bedner, E. & Smolewski, P. Flow cytometry in analysis of cell cycle and apoptosis. Semin. Hematol. 38, 179–193 (2001).

    Article  CAS  Google Scholar 

  9. Telford, W.G., King, L.E. & Fraker, P.J. Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J. Immunol. Methods 172, 1–16 (1994).

    Article  CAS  Google Scholar 

  10. Wallen, C.A., Higashikubo, R. & Dethlefsen, L.A. Comparison of two flow cytometric assays for cellular RNA—acridine orange and propidium iodide. Cytometry 3, 155–160 (1982).

    Article  CAS  Google Scholar 

  11. Pollack, A. & Ciancio, G. Cell cycle phase-specific analysis of cell viability using Hoechst 33342 and propidium iodide after ethanol preservation. Methods Cell. Biol. 33, 19–24 (1990).

    Article  CAS  Google Scholar 

  12. Ormerod, M.G., Collins, M.K., Rodriguez-Tarduchy, G. & Robertson, D. Apoptosis in interleukin-3-dependent haemopoietic cells. Quantification by two flow cytometric methods. J. Immunol. Methods 153, 57–65 (1992).

    Article  CAS  Google Scholar 

  13. Schmid, I., Uittenbogaart, C.H., Keld, B. & Giorgi, J.V. A rapid method for measuring apoptosis and dual-color immunofluorescence by single laser flow cytometry. J. Immunol. Methods 170, 145–157 (1994).

    Article  CAS  Google Scholar 

  14. Wyllie, A.H., Morris, R.G., Smith, A.L. & Dunlop, D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67–77 (1984).

    Article  CAS  Google Scholar 

  15. Gavrieli, Y., Sherman, Y. & Ben-Sasson, S.A. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501 (1992).

    Article  CAS  Google Scholar 

  16. Telford, W.G., Komoriya, A. & Packard, B.Z. Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry. Cytometry 47, 81–88 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC, to CR), Milan, Italy.

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  1. Department of Clinical and Experimental Medicine, School of Medicine, University of Perugia, and Foundation IBiT, 06122, Perugia, Italy

    Carlo Riccardi & Ildo Nicoletti

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  1. Carlo Riccardi
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  2. Ildo Nicoletti
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Correspondence to Carlo Riccardi.

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Riccardi, C., Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1, 1458–1461 (2006). https://doi.org/10.1038/nprot.2006.238

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