Measurement of DNA damage associated with apoptosis by laser scanning cytometry

Background: Phosphatidylserine (PS) binding by annexin V (AV) is an early m embrane marker of apoptosis. Using laser scanning cytometry (LSC) and the c omet assay, we showed that the DNA of AV(+) cells is so highly fragmented t hat 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 D NA damage associated with apoptosis. The LSC was used to measure both AV fl uorescence and DNA damage on the same jurkat cells following treatment with anti-Fas. The data from both sets of measurements were merged, allowing di rect correlation of membrane and nuclear markers of cell death. Results: AV+ cells had significant DNA damage determined by the ratio betwe en nuclear DNA and peripheral (migrated) DNA. Cells in the early and late s tages of apoptosis could be discriminated on the basis of DNA content. In a ddition, it was possible to distinguish between apoptotic and necrotic cell s in the AV(+) propidium iodide-positive population based on DNA content an d DNA damage. The addition of specific inhibitors for caspases-8, 9, and 3 blocked both PS externalization and DNA fragmentation, indicating these eve nts are downstream from caspase activation. Conclusions: This technique allows accurate distinction between apoptotic a nd necrotic cells and cytometric grading of apoptosis. Cytometry 45:180-186 , 2001. (C) 2001 Wiley-Liss, Inc.