Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Dec 1;25(23):4855–4857. doi: 10.1093/nar/25.23.4855

A rapid, quantitative and inexpensive method for detecting apoptosis by flow cytometry in transiently transfected cells.

G M Lamm 1, P Steinlein 1, M Cotten 1, G Christofori 1
PMCID: PMC147110  PMID: 9365268

Abstract

We describe a rapid and quantitative flow cytometric method for determining the apoptotic or anti-apoptotic potential of a gene in various cell types. A plasmid carrying green fluorescent protein (GFP) is co-transfected with an expression vector encoding the gene of interest. Subsequently cells are stained with propidium iodide and, utilising flow cytometry, transfected, GFP-expressing single cells are detected and apoptotic cells in this population are identified by their DNA content of <2 N. The method detects apoptosis as reliably as established methods using in situ nick-end labelling but is faster, easier and less expensive.

Full Text

The Full Text of this article is available as a PDF (44.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker A., Saltik M., Lehrmann H., Killisch I., Mautner V., Lamm G., Christofori G., Cotten M. Polyethylenimine (PEI) is a simple, inexpensive and effective reagent for condensing and linking plasmid DNA to adenovirus for gene delivery. Gene Ther. 1997 Aug;4(8):773–782. doi: 10.1038/sj.gt.3300471. [DOI] [PubMed] [Google Scholar]
  2. Chinnaiyan A. M., Dixit V. M. The cell-death machine. Curr Biol. 1996 May 1;6(5):555–562. doi: 10.1016/s0960-9822(02)00541-9. [DOI] [PubMed] [Google Scholar]
  3. Cormack B. P., Valdivia R. H., Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene. 1996;173(1 Spec No):33–38. doi: 10.1016/0378-1119(95)00685-0. [DOI] [PubMed] [Google Scholar]
  4. Darzynkiewicz Z., Bruno S., Del Bino G., Gorczyca W., Hotz M. A., Lassota P., Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry. 1992;13(8):795–808. doi: 10.1002/cyto.990130802. [DOI] [PubMed] [Google Scholar]
  5. Eldadah B. A., Yakovlev A. G., Faden A. I. A new approach for the electrophoretic detection of apoptosis. Nucleic Acids Res. 1996 Oct 15;24(20):4092–4093. doi: 10.1093/nar/24.20.4092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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. 1992 Nov;119(3):493–501. doi: 10.1083/jcb.119.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanahan D. Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature. 1985 May 9;315(6015):115–122. doi: 10.1038/315115a0. [DOI] [PubMed] [Google Scholar]
  8. Ioannou Y. A., Chen F. W. Quantitation of DNA fragmentation in apoptosis. Nucleic Acids Res. 1996 Mar 1;24(5):992–993. doi: 10.1093/nar/24.5.992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jacobsen M. D., Weil M., Raff M. C. Role of Ced-3/ICE-family proteases in staurosporine-induced programmed cell death. J Cell Biol. 1996 Jun;133(5):1041–1051. doi: 10.1083/jcb.133.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kerr J. F., Gobé G. C., Winterford C. M., Harmon B. V. Anatomical methods in cell death. Methods Cell Biol. 1995;46:1–27. doi: 10.1016/s0091-679x(08)61921-4. [DOI] [PubMed] [Google Scholar]
  11. Koopman G., Reutelingsperger C. P., Kuijten G. A., Keehnen R. M., Pals S. T., van Oers M. H. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood. 1994 Sep 1;84(5):1415–1420. [PubMed] [Google Scholar]
  12. Korsmeyer S. J. Regulators of cell death. Trends Genet. 1995 Mar;11(3):101–105. doi: 10.1016/S0168-9525(00)89010-1. [DOI] [PubMed] [Google Scholar]
  13. McGahon A. J., Martin S. J., Bissonnette R. P., Mahboubi A., Shi Y., Mogil R. J., Nishioka W. K., Green D. R. The end of the (cell) line: methods for the study of apoptosis in vitro. Methods Cell Biol. 1995;46:153–185. doi: 10.1016/s0091-679x(08)61929-9. [DOI] [PubMed] [Google Scholar]
  14. 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. 1991 Jun 3;139(2):271–279. doi: 10.1016/0022-1759(91)90198-o. [DOI] [PubMed] [Google Scholar]
  15. Radvanyi F., Christgau S., Baekkeskov S., Jolicoeur C., Hanahan D. Pancreatic beta cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines. Mol Cell Biol. 1993 Jul;13(7):4223–4232. doi: 10.1128/mcb.13.7.4223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Raff M. C., Barres B. A., Burne J. F., Coles H. S., Ishizaki Y., Jacobson M. D. Programmed cell death and the control of cell survival: lessons from the nervous system. Science. 1993 Oct 29;262(5134):695–700. doi: 10.1126/science.8235590. [DOI] [PubMed] [Google Scholar]
  17. Salgame P., Varadhachary A. S., Primiano L. L., Fincke J. E., Muller S., Monestier M. An ELISA for detection of apoptosis. Nucleic Acids Res. 1997 Feb 1;25(3):680–681. doi: 10.1093/nar/25.3.680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Telford W. G., King L. E., Fraker P. J. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry. Cytometry. 1992;13(2):137–143. doi: 10.1002/cyto.990130205. [DOI] [PubMed] [Google Scholar]
  19. Telford W. G., King L. E., Fraker P. J. Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. Cell Prolif. 1991 Sep;24(5):447–459. doi: 10.1111/j.1365-2184.1991.tb01173.x. [DOI] [PubMed] [Google Scholar]
  20. Vaux D. L., Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2239–2244. doi: 10.1073/pnas.93.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. White E., Cipriani R. Role of adenovirus E1B proteins in transformation: altered organization of intermediate filaments in transformed cells that express the 19-kilodalton protein. Mol Cell Biol. 1990 Jan;10(1):120–130. doi: 10.1128/mcb.10.1.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. White E., Cipriani R., Sabbatini P., Denton A. Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins. J Virol. 1991 Jun;65(6):2968–2978. doi: 10.1128/jvi.65.6.2968-2978.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. White E. Life, death, and the pursuit of apoptosis. Genes Dev. 1996 Jan 1;10(1):1–15. doi: 10.1101/gad.10.1.1. [DOI] [PubMed] [Google Scholar]
  24. Wyllie A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature. 1980 Apr 10;284(5756):555–556. doi: 10.1038/284555a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES