Apoptosis

, Volume 3, Issue 2, pp 115–121 | Cite as

Early Features of Apoptosis Detected by Four Different Flow Cytometry Assays

  • R. Overbeeke
  • H. Steffens-Nakken
  • I. Vermes
  • C. Reutelingsperger
  • C. Haanen

Abstract

The objective of this study was to investigate the sensitivity, specificity and reproducibility of some frequently used apoptosis assays. The degree of apoptosis was tested in two T-lymphoblastoid cell lines, HSB and Jurkat, in which apoptosis was induced by ionizing radiation. HSB and Jurkat samples were taken before, and 0, 2, 4, 6, 8 and 24 h after irradiation with 6 and 10 Gray, or with 10 and 14 Gray, respectively. Four frequently used flow cytometric techniques were evaluated: (i) Annexin V/Propidium Iodide assay, detecting the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, simultaneously with preservation of the membrane integrity; (ii) Terminal deoxynucleotidyl Transferase (TdT) Uridine triphosphate (UTP) nick end labelling (TUNEL), revealing the presence of DNA strand breaks; (iii) DNA-flow cytometry, measuring DNA-stainability (DNA-fragmentation assay) and (iv) Phycoerythrin-labelled (PE) Apo2.7-assay, a monoclonal antibody against 7A6 antigen, a protein, which becomes exposed upon the mitochondrial membrane during apoptosis. As a general standard for identifying that apoptosis had occurred, the cells were assessed for the presence of DNA-laddering on agar gel electrophoresis and by demonstration of characteristic cell morphology. Results were as follows: Fluorescein Isothiocyanate (FITC)-labelled Annexin V/Propidium iodide flow cytometry appeared to be the most sensitive, the most specific and the most user-friendly test for measurement of apoptosis of cells in culture conditions in suspension. The expression of 7A6 antigen on the mitochondrial membrane appeared to be not specific for apoptotic cell death.

7A6-antigen Annexin V apoptosis DNA fragmentation phosphatidyl serine 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Martin SJ, Reutelingsperger CPM, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med 1995, 182: 1545–1556.Google Scholar
  2. 2.
    Koopman G, Reutlingsperger CPM, Kuijten GAM, Kechnen RMJ, Pals ST, Van Oers MHJ. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: 1415–1420.Google Scholar
  3. 3.
    Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger CPM. A novel assay for apoptosis: Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using Fluorescein labeled Annexin V. J Immunol Meth 1995; 184: 39–51.Google Scholar
  4. 4.
    Zhang C, Ao Z, Seth A, Schlossman SF. A mitochondrial membrane protein defined by an novel monoclonal antibody is preferentially detected in apoptotic cells. J Immunol 1996; 157: 3980–3987.Google Scholar
  5. 5.
    Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Meth 1991; 139: 271–279.Google Scholar
  6. 6.
    Meyaard L, Otto SA, Jonker RR, Mijnster MJ, Keet RPM, Miedema F. Programmed death of T cells in HIV-1 infection. Science 1992; 257: 217–219.Google Scholar
  7. 7.
    Gavrieli Y, Sherman Y, Ben Sasson SA. Identification of programmed cell death in situ specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493–501.Google Scholar
  8. 8.
    Gorczyca W, Bruno S, Darzynkiewicz RJ, Gong J, Darzynckiewicz Z. DNA strand breaks occurring during apoptosis: Their early in situ detection by terminal deoxynucleotidyl Transferase and nick translation assays and prevention by serine protease inhibitors. nt J Oncol 1992; 1: 639–648.Google Scholar
  9. 9.
    Wolfe JT, Pringle JH, Cohen GM. Assays for the measurement of DNA fragmentation during apoptosis. In: Cotter TG, Martin SJ, eds. Techniques in Apoptosis. A User's Guide. London: Portland Press Ltd 1996: 51–69.Google Scholar
  10. 10.
    Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 11: 1215.Google Scholar

Copyright information

© Rapid Science Ltd 1998

Authors and Affiliations

  • R. Overbeeke
    • 1
  • H. Steffens-Nakken
    • 1
  • I. Vermes
    • 1
  • C. Reutelingsperger
    • 2
  • C. Haanen
    • 1
  1. 1.Department of Clinical Chemistry, Medical Spectrum Twente, Hospital Group, EnschedeUniversity MaastrichtThe Netherlands
  2. 2.Department of BiochemistryUniversity MaastrichtThe Netherlands

Personalised recommendations