Comet assay and DNA flow cytometry analysis of staurosporine-induced apoptosis

Background: The ability of the comet assay to quantify DNA strand breaks an d alkali labile sites has been widely demonstrated. In this study, this ass ay was tested for its ability to identify DNA fragmentation occurring durin g apoptosis in comparison with standard DNA flow cytometry analysis. Methods: Staurosporine-induced apoptosis in CHO cells is an adequate model to study a rapid time- and dose-dependent appearance of this process. Results: Nuclear staining with DAPI confirmed the induction of apoptosis wi th a typical chromatin condensation and fragmentation. Analysis of propidiu m-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G (1) peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatm ent occurred earlier than the detection of apoptotic cells by now cytometry . However, HDC were missed when the DNA fragmentation was too high, prevent ing accurate quantification of late apoptotic cells. Conclusions: The comet assay is more sensitive than standard DNA flow cytom etry to detect early DNA fragmentation events occurring during apoptosis. H owever, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at laterstages. Cytometry 36: 117-122, 1999. (C) 1999 Wiley-Liss, Inc.