NeutralCometAssay

NeutralCometAssay, also called the neutral comet assay or neutral single-cell gel electrophoresis, is a variant of the comet assay focused on detecting DNA double-strand breaks in individual cells. It uses neutral pH conditions during lysis and electrophoresis to preserve double-strand breaks and to differentiate them from other forms of damage.

In typical protocols, cells are embedded in a thin layer of agarose on slides, lysed to remove membranes and proteins, and then subjected to electrophoresis in a neutral buffer. After staining with a DNA-binding dye, the DNA forms a comet-like image under fluorescence microscopy, with a head representing intact DNA and a tail reflecting fragmented DNA. Quantification usually involves tail length, percentage of DNA in the tail, or an olive tail moment, which correlates with the amount of double-strand breaks per cell.

Applications of the neutral assay include assessment of genotoxic effects from radiation, chemicals, and environmental exposures, as well as studying DNA repair kinetics and the impact of protective agents in various cell types, including human lymphocytes and cultured mammalian cells.

Interpretation of results centers on the notion that more extensive tailing indicates more double-strand breaks. The neutral assay is more specific for DSBs than the alkaline version but is generally less sensitive and can be more influenced by chromatin structure and cell cycle. Standardization and careful handling are important to minimize artifactual breaks and to enable meaningful comparisons across studies.