Measurement of DNA damage associated with apoptosis by laser scanning cytometry

Zsolt Bacso, James F. Eliason

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Background: Phosphatidylserine (PS) binding by annexin V (AV11) is an early membrane marker of apoptosis. Using laser scanning cytometry (LSC) and the comet assay, we showed that the DNA of AV+ cells is so highly fragmented that it cannot be quantified by the comet assay (Bacso et al.: Cancer Res 60:4623-8, 2000). Methods: The "halo" assay was used instead of the comet assay to quantify DNA damage associated with apoptosis. The LSC was used to measure both AV fluorescence and DNA damage on the same Jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing direct correlation of membrane and nuclear markers of cell death. Results: AV+ cells had significant DNA damage determined by the ratio between nuclear DNA and peripheral (migrated) DNA. Cells in the early and late stages of apoptosis could be discriminated on the basis of DNA content. In addition, it was possible to distinguish between apoptotic and necrotic cells in the AV+ propidium iodide-positive population based on DNA content and DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these events are down-stream from caspase activation. Conclusions: This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.

Original languageEnglish
Pages (from-to)180-186
Number of pages7
JournalCytometry
Volume45
Issue number3
DOIs
Publication statusPublished - Jan 1 2001

Keywords

  • Annexin V
  • Apoptosis
  • Caspase inhibitors
  • DNA damage
  • Laser scanning cytometer

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

Fingerprint Dive into the research topics of 'Measurement of DNA damage associated with apoptosis by laser scanning cytometry'. Together they form a unique fingerprint.

  • Cite this