The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment.
Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.
Ashby
J
J
Zeneca Central Toxicology Laboratory, Nr Macclesfield, Cheshire, UK.
Tinwell
H
H
Lefevre
P A
PA
Browne
M A
MA
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000187:drug effects; D004249:DNA Damage; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004128:Dimethylnitrosamine / Q000494:pharmacology; D004586:Electrophoresis / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000166:cytology; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D011919:Rats, Inbred Strains
D004247:DNA; D004128:Dimethylnitrosamine; D004121:Dimethyl Sulfoxide