Early detection of staurosporine-induced apoptosis by comet and annexin V assays

Abstract

Comet, TUNEL, and annexin V assays were used to identify DNA fragmentation and plasma membrane alterations occurring during staurosporine-induced apoptosis in Chinese hamster ovary cells. TUNEL assay detected apoptotic cells after 6 h treatment. The occurrence of annexin V immunofluorescence staining after 1 h treatment confirms that exposure of phosphatidylserine (PS) residues is an early biochemical feature of apoptosis. According to intensity, three annexin staining patterns were distinguished, related to different steps in the apoptotic process. The detection of highly damaged cells by the comet assay after 3 h treatment occurred earlier than the detection of DNA modifications by the TUNEL assay, but later than the exposure of PS residues. However, late apoptotic cells, otherwise characterized by plasma membrane disruption and high annexin V staining, were not detected by the comet assay. In this case, comet assay modified by omitting electrophoresis (halo assay) was more sensitive for an accurate quantification of the apoptotic fraction.

Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

“…The alkaline single cell gel electrophoresis assay (Comet assay) was developed to assess DNA fragmentation typical of toxic DNA damage and of early-stage apoptosis (14,15). This assay, when electrophoresis is performed at pH Ն13, provides a sensitive means of analyzing alkali-labile sites and singleand double-strand DNA breaks in small number of cells.…”

Amifostine (WR-2721) selective protection against melphalan genotoxicity

“…34,35 The terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) assay has also proved to be a sensitive and reliable method for measuring DNA strand breaks. However, the detection of highly damaged cells by the Comet assay occurred earlier than the detection of DNA modifications by the TUNEL assay 36 and more sensitively. 37 SCGE allowed a rapid analysis of genotoxic damage in individual nuclei such as flow cytometry, and a significant strong correlation was shown 38,39 with a higher sensitivity.…”

Single‐cell gel electrophoresis assay monitors precise kinetics of DNA fragmentation induced during programmed cell death

“…Comets from the apoptotic bodies are generally Unwinding in 30 mM NaOH, 2 mM EDTA for 20 min at RT; electrophoresis in 30 mM NaOH for 4 min at 0. shorter than 20 m, although such comets were rarely observed in this study. The lysis and electrophoresis conditions used in the present study were different from those used in most of the comet assay methods reported for detecting DNA fragmentation (7,11,13,15,17,18). Whereas the present method mainly used anionic detergents and a neutral lysis condition (pH 9.5) followed by electrophoresis at nearly neutral or low alkaline pH (8.3), previous studies have generally used high-salt lysis at low alkaline pH followed by neutral or alkaline electrophoresis.…”

“…Alkaline and neutral versions of the assay have been developed that predominantly detect singleand double-strand breaks, respectively (5,6). Due to its ability to detect DNA breaks, comet assay also has been used in the detection of DNA fragmentation during apoptosis (7)(8)(9)(10)(11)(12). The neutral version of the assay is preferred for this purpose due to its specificity for double-strand breaks (13).…”