TY - JOUR
T1 - Technical Updates to the Comet Assay in Vivo for Assessing DNA Damage in Zebrafish Embryos from Fresh and Frozen Cell Suspensions
AU - Martins, Carla F.
AU - Costa, Pedro M.
N1 - info:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F120030%2F2016/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04378%2F2020/PT#
PY - 2020/6/1
Y1 - 2020/6/1
N2 - The Single-Cell Gel Electrophoresis, simply known as the Comet assay, is a sensitive and quick technique used to quantitate DNA damage, widely used to assess the effects of genotoxicants and mutagens in animal cells. Still, performing the assay on peripheral or cultured cells is far more expeditious and cost effective than solid tissue, especially from small biological model like the zebrafish embryo. The current work describes and validates a highly cost-effective protocol of the updated Comet assay designed for zebrafish embryos. Compared with the few previous applications of the Comet assay on this biological model, the present method successfully simplifies the process of cell harvesting and resuspending, producing a much higher yield of viable nucleoids with reduced basal DNA damage, even from a small number of embryos, and compatible with scoring with safe fluorescent dyes. Additionally, the protocol can be just as easily performed on freshly harvested cells of cells frozen in dimethyl sulfoxide (DMSO)-containing physiological buffer, without a significant increase of DNA damage, which is another highly relevant update, especially for researchers handling high numbers of samples.
AB - The Single-Cell Gel Electrophoresis, simply known as the Comet assay, is a sensitive and quick technique used to quantitate DNA damage, widely used to assess the effects of genotoxicants and mutagens in animal cells. Still, performing the assay on peripheral or cultured cells is far more expeditious and cost effective than solid tissue, especially from small biological model like the zebrafish embryo. The current work describes and validates a highly cost-effective protocol of the updated Comet assay designed for zebrafish embryos. Compared with the few previous applications of the Comet assay on this biological model, the present method successfully simplifies the process of cell harvesting and resuspending, producing a much higher yield of viable nucleoids with reduced basal DNA damage, even from a small number of embryos, and compatible with scoring with safe fluorescent dyes. Additionally, the protocol can be just as easily performed on freshly harvested cells of cells frozen in dimethyl sulfoxide (DMSO)-containing physiological buffer, without a significant increase of DNA damage, which is another highly relevant update, especially for researchers handling high numbers of samples.
KW - comet metrics
KW - Danio rerio
KW - genotoxicity
KW - protocol
KW - single-cell gel electrophoresis
UR - http://www.scopus.com/inward/record.url?scp=85088617390&partnerID=8YFLogxK
U2 - 10.1089/zeb.2020.1857
DO - 10.1089/zeb.2020.1857
M3 - Article
C2 - 32240067
AN - SCOPUS:85088617390
SN - 1545-8547
VL - 17
SP - 220
EP - 228
JO - Zebrafish
JF - Zebrafish
IS - 3
ER -