BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during 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 with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.
BACKGROUND: The ability of the comet assay to quantify DNA strand breaks and alkali labile sites has been widely demonstrated. In this study, this assay was tested for its ability to identify DNA fragmentation occurring during 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 with a typical chromatin condensation and fragmentation. Analysis of propidium-iodide- (PI) stained DNA by flow cytometry showed the presence of a pre-G1 peak, characteristic of apoptotic cells, 6 h after drug treatment. The detection of highly damaged cells (HDC) by the comet assay after 3 h treatment occurred earlier than the detection of apoptotic cells by flow cytometry. However, HDC were missed when the DNA fragmentation was too high, preventing accurate quantification of late apoptotic cells. CONCLUSIONS: The comet assay is more sensitive than standard DNA flow cytometry to detect early DNA fragmentation events occurring during apoptosis. However, the comet assay modified by omitting electrophoresis was necessary to quantify apoptotic fraction at later stages.
Authors: Adriana R Cruz; Meagan W Moore; Carson J La Vake; Christian H Eggers; Juan C Salazar; Justin D Radolf Journal: Infect Immun Date: 2007-10-15 Impact factor: 3.441