Single-Cell Gel Electrophoresis (Comet Assay) and Comet Scoring

The comet assay, also known as the single-cell gel electrophoresis assay, is an easy and sensitive technique that enables a quantitative assessment of the effects of DNA damage or apoptosis induction and the underlying mechanisms involved in different target cell types. The assay has also been utilized in studies regarding DNA repair.

The comet assay is based on cell migration through electrophoresis and the quantitative analysis of populations of targeted cells. In general, cells are suspended in an agarose gel that is layered onto a microscope slide. Electrophoresis causes the migration of unwound or fragmented DNA out of the cell’s nucleus, which results in a characteristic, comet-like shape and appearance that can be easily seen microscopically with fluorescent staining of the DNA. The measurement and quantitative analysis of the fluorescent intensities revealed by the cells gives masseurs of cellular DNA content distribution and shows damage if present. The single-cell gel electrophoresis (comet assay) was carried out to characterize and quantify the degree of DNA damage under an alkaline condition as described by others but with slight modifications. ¹⁶ Approximately 1-2 × 10⁴ leukocytes were combined with 100 μL of melted agarose (LMA), and 75 μL of this solution was immediately pipetted onto the comet slide (Trevigen, USA). Next, the slides were immersed in a freshly prepared cold lysing solution (2.5 M NaCl, 100 mM Na₂ EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO) for a minimum of 1 h at 4°C. The slides were then placed in a horizontal electrophoresis apparatus filled with freshly prepared alkaline solution (pH ≥ 13). The slides were immersed in the lysing solution for 20 min before electrophoreses was performed at 1 volt/cm for 20 min. Next, the slides were rinsed three times for 5 min in the washing buffer (0.4 M of Tris at pH 7.4) and immersed in absolute ethanol for 5 min. After air drying, the slides were stained with ethidium bromide (0.5 μg/mL), covered, and analyzed.

Each sample was processed in duplicate to avoid processing errors. The slides were then examined under a fluorescence microscope for comet scoring. The extent of the DNA damage was measured quantitatively using a comet analysis system (Loats, USA) based on extended dynamic range imaging (EDRI) technology, which expressed the comet tail moment automatically. Parallel to each experiment, a series of negative controls were carried out to determine any non-specific formation or reduction in the comet. The percentage of the comet cells were calculated as follows:

The means and standard deviations (SDs) for the tail moment values for all images in each group that was generated using EDRI technology were calculated.