Neutral Comet Assay

Abstract

[Abstract] The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a "comet", while undamaged DNA remained in the head of the "comet". The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair. Materials and Reagents 1. Cells to analyze 2. Low Melting Point (LMP) Agarose (Sigma-Aldrich, catalog number: A9414) 3. Seakem ® Agarose (Ozyme, catalog number: LON50004) 4. PBS (Ca 2+ and Mg 2+-free phosphate-buffered saline) 5. 5 N NaOH 6. 0.5 M EDTA disodium salt solution (pH 8) 7. Trisma base 8. Triton X-100 9. N-Lauroylsarcosine (Sigma-Aldrich, catalog number: L5125) 10. Dimethylsulphoxide (DMSO) 11. Absolute ethanol 12. Ethidium bromide (10 mg/ml) 13. Trypsin/EDTA 14. Sodium acetate 15. Lysis solution (see Recipes) 16. Electrophoresis solution (see Recipes)