The in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet
Assay is a method measuring DNA strand breaks in eukaryotic cells.Each treated group
is composed of a minimum of 5 animals of one sex (or of each sex as appropriate).
A positive and a vehicle control group are also used. Administration of the treatment
consists of daily doses over duration of 2 days or more, ensuring the test chemical
reaches the target tissue which can be the liver, the kidney or other tissues if justified.Tissues
of interest are dissected and single cells/nuclei suspensions are prepared and embedded
in agarose on slides. Cells/nuclei are treated with lysis buffer to remove cellular
and/or nuclear membranes. The nuclear DNA in the agar is then subjected to electrophoresis
at high pH. This results in structures resembling comets which by using suitable fluorescent
stain, can be observed by fluorescent microscopy. Based on their size DNA fragments
migrate away from the head to the tail, and the intensity of the comet tail relative
to the total intensity (head plus tail) reflects the amount of DNA breakage.