Isolation of leukocytes from frozen buffy coat for comet assay analysis of DNA damage

Publication date: Available online 31 March 2019Source: Mutation Research/Genetic Toxicology and Environmental MutagenesisAuthor(s): Siv Kjølsrud Bøhn, Vaineta Vebraite, Sergey Shaposhnikov, Andrew R. CollinsAbstractFrozen buffy coat fractions are often stored in human biomonitoring trials but their use for biomarker purposes has been limited. The purpose of the current study was to study whether frozen buffy coats can be used to monitor DNA damage levels.EDTA blood samples were provided from 9 healthy, non-smoking female volunteers, aged 26-48. Pre-existing DNA damage (strand breaks and oxidised purines) was measured with the comet assay in thawed resuspended buffy coat samples and washed leukocytes from these buffy coats, as well as resistance to DNA damage induced exogenously by H2O2 in the latter, and compared with damage measured in peripheral blood mononuclear cells isolated from fresh blood using percoll gradient centrifugation.Basal DNA damage levels (strand breaks) were significantly higher in the leukocytes isolated from frozen buffy coats in the untreated samples compared with peripheral blood mononuclear cells. However, the levels of strand breaks were still low (<4% tail DNA), indicating that little damage is caused by freezing or processing. Base oxidation was significantly higher in isolated buffy coat leukocytes than in peripheral blood mononuclear cells from fresh blood, but showed a good correlation (r = 0.67) between the two cell types. The correl...
Source: Mutation Research Genetic Toxicology and Environmental Mutagenesis - Category: Genetics & Stem Cells Source Type: research