Measurement of DNA damage associated with apoptosis by laser scanning cytometry.
BACKGROUND
Phosphatidylserine (PS) binding by annexin V (AV) 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 downstream from caspase activation.


CONCLUSIONS
This technique allows accurate distinction between apoptotic and necrotic cells and cytometric grading of apoptosis.
Bacso
Z
Z
Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA. bacso@jaguar.dote.hu
Eliason
J F
JF
D017304:Annexin A5 / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D004247:DNA / Q000032:analysis; D004249:DNA Damage / Q000502:physiology*; D053938:DNA Fragmentation / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D009336:Necrosis; D011419:Propidium / Q000032:analysis
D017304:Annexin A5; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D061945:Caspase Inhibitors; C411023:anti-Fas monoclonal antibody; D011419:Propidium; D004247:DNA; D020169:Caspases