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

Abstract

Background: Single-cell gel electrophoresis, or the comet assay, a technique widely used for DNA damage analysis, has been used recently for detecting DNA fragmentation in cells undergoing apoptosis. However, the number of variants of this assay used thus far primarily detected the late stages of DNA fragmentation. Therefore, monitoring the progression of DNA fragmentation, which could greatly improve the analysis of cell death induction and progression at the single-cell level, has not been possible with this assay. Methods: In the present study, a modification of the original neutral comet assay developed by Ostling and Johanson (Biochem Biophys Res Commun 123:291-298, 1984) was used to detect various stages of DNA fragmentation. This assay involves cell lysis with anionic detergents at nearly neutral pH (9.5) and does not include high salt concentration, unlike most other published methods. BMG-1 human glioma cells were induced to undergo programmed cell death by treating with a large dose (100 M) of etoposide, and comets were prepared after different durations (1-24 h) of treatment. Results: In contrast to results of previously published studies, comets with different shapes reflecting progressive stages of DNA fragmentation were observed. Of these, six distinct shapes were identified and divided into

The nuclear actin‐related protein Act3p/Arp4 influences yeast cell shape and bulk chromatin organization

“…We have chosen the most often used parameters, comet length (C length ) and TM Figure 3C. These parameters represent DNA liberated from the head of the comet and are proportional to the degree of chromatin relaxation [Tice et al, 2000;Chandna, 2004]. Both parameters are representative and in the case of nuclease treatment definitely revealed changes in the chromatin organization of the eukaryotic nucleus.…”

An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

“…The extensive DNA fragmentation induced during apoptosis can be detected using techniques such as DNA ladder assay (agarose gel electrophoresis), terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL assay) and comet assay (Otsuki 2000;Chandna 2004). Out of these methods, DNA fragmentation assay using agarose gel electrophoresis is the most frequent technique used for the detection of apoptosis and can easily discriminate between apoptotic and non-apoptotic (necrotic) modes of cell death, as in most cases the inter-nucleosomal cleavage of genomic DNA yielding the characteristic DNA ladder is a molecular hallmark of apoptotic cells (Kerr et al 1972;Pandey et al 1994).…”

GHRH antagonist causes DNA damage leading to p21 mediated cell cycle arrest and apoptosis in human colon cancer cells

“…S4). The number of apoptotic comets (Type-A, -B, -C), 10 observed following the treatment with JMR-132 is shown in Figure 3A and B. In accordance with the pro-apoptotic alterations in Bax/BCL-2 levels, comet assay detected DNA fragmentation in more than 25% of cells as early as 1 h, which increased with the progression of fragmentation (~75% type-C comets at 4 h), depicting terminal stage of DNA fragmentation.…”

“…Neutral comet assay with its simple modifications described earlier was used to study DNA damage. 10 Apoptotic DNA fragmentation yields neutral comets with progressively increasing DNA content and area within the tail region and a correspondingly reducing head size. Based on this progression, apoptotic comets were divided into three basic shapes.…”