The Comet Moment as a Measure of DNA Damage in the Comet Assay

Abstract

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

Reduction in drug-induced DNA double-strand breaks associated with β1 integrin–mediated adhesion correlates with drug resistance in U937 cells

“…The images were captured on a fluorescent microscope (Vysis, Downers Grove, IL) and quantified by using Imagequant software (Molecular Dynamics, Sunnyvale, Ca). The comet moment was calculated by using the following equation described by Kent et al 12 : comet moment ϭ ⌺ 0Ϫn ((intensity of DNA at distance X) ϫ (distance))/intensity of total DNA.…”

“…After 2 hours of adhesion, cells were exposed to various concentrations of drug for 1 hour. Subsequently, 5000 cells were placed in a microcentrifuge tube containing 1 mL cold PBS, and the neutral comet assay was done as described by Kent et al 12 Briefly, cells were centrifuged and resuspended in 500 L cold PBS, and 1.5 mL 1% agarose was added to each sample. The agarose-cell suspension was gently layered on a frosted-glass microscope slide, allowed to solidify for 5 minutes, and then placed immediately in ice-cold lysis buffer containing 30 mM disodium ethylenediamine tetraacetic acid (EDTA, pH 8.0), 0.5% sodium dodecyl sulfate (SDS), and 0.25 mg/mL proteinase K (Fisher Scientific, Norcross, GA).…”

Miniaturizing the comet assay with 3D vertical comets

“…Descriptions of all kinds of parameters used in comet quantification can be found in the literature (7,8,18) and was adapted for 3D data sets. Parameters used were tail length, head:tail ratio, %tail, comet moment, and comet inertia.…”

“…This system performs automatic cell recognition and measures the DNA damage as a decrease in integrated fluorescence in the comet head compared with the integrated fluorescence of DNA in normal undamaged cell nuclei; 1,000 comets can be scanned in 30 min. The computation of more sensitive parameters (7,8) relies on the rescanning of the microscope slide for comets belonging to the sub-G1 region of the histogram and conventional image processing. For both methodologies described, a very thin layer of cell containing gel and the use of lower resolution objectives (20ϫ) are crucial for appropriate focusing.…”

“…Image analysis has widely become the procedure of choice due to its advanced quantitative approach. Several parameters based on the distribution of DNA in the comet head and tail can be used to quantify DNA damage (7,8). One of these parameters is known as the comet moment and is regarded as the most sensitive because it uses the mass and distance of DNA transported into an electrical field (8).…”