The CometAssay®, or single cell gel electrophoresis assay, provides a simple and effective method for evaluating DNA damage. The principle of the assay is based upon the ability of denatured cleaved DNA fragments to migrate out of the cell under the influence of an electric potential (the "comet tail"), whereas undamaged supercoiled DNA remains within the confines of the cell membrane (the "comet head") when a current is applied.
This method involves the immobilization of cells in a bed of low melting point agarose. After treatment with alkali to denature the DNA and hydrolyse sites of damage, the samples are submitted to electrophoresis. Samples are subsequently stained with a fluorescent DNA-intercalating dye or silver stain and visualized using the appropriate microscope.
CometAssay – Silver
Assay kit for unfixed cells and fresh tissue samples designed for light microscopy (50 Tests) (Catalog # 4251-050-K).
Visualization of DNA Damage using the Comet Assay and Silver Staining. Murin WEHI 7.1 lymphoma cells were treated with potassium permanganate and processed using the CometAssay - Silver Staining Kit (Catalog # 4251-050-K) under alkali conditions. Following electrophoresis, samples were fixed in cold ethanol and air dried. After drying, the slides were stained as recommended in the kit instructions and analyzed by microscopy. DNA damage is indicated by a "comet tail" shape and the overall migration pattern of the DNA.
CometAssay - SYBR
Assay kit for unfixed cells and fresh tissue samples designed for fluorescence microscopy (50 Tests) (Catalog # 4250-050-K).
Visualization of DNA Damage using the CometAssay - SYBR Kit. Murin WEHI 7.1 lymphoma cells were trated with potassium permanganate and DNA fragmentation was visualized using the CometAssay - SYBR Kit (Catalog # 4250-050-K) under alkali conditions. Following electrophoresis the samples were fixed and dried. The slides were subsequently stained following the instructions provided in the kit, and the cells were analyzed by microscopy. Undamaged DNA does not migrated far from the origin, while damaged DNA appears as a fluorescent "comet tail".