New Application of the Comet Assay

The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be used to generate comets in subnuclear units, such as chromosomes. The new technique is based on the chromosome isolation protocols currently used for whole chromosome mounting in electron microscopy, coupled to the alkaline variant of the comet assay, to detect DNA damage. The results show that migrant DNA fragments can be visualized in whole nuclei and isolated chromosomes and that they exhibit patterns of DNA migration that depend on the level of DNA damage produced. This protocol has great potential for the highly reproducible study of DNA damage and repair in specific chromosomal domains.

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Effect of cryopreservation on DNA damage and DNA repair activity in human blood samples in the comet assay

Archives of Toxicology ◽

10.1007/s00204-021-03012-4 ◽

2021 ◽

Author(s):

Ezgi Eyluel Bankoglu ◽

Franzisca Stipp ◽

Johanna Gerber ◽

Florian Seyfried ◽

August Heidland ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Dna Damage ◽

Comet Assay ◽

Human Blood ◽

Ex Vivo ◽

Human Biomonitoring ◽

Dna Damage And Repair ◽

Blood Samples ◽

Repair Activity ◽

Human Blood Samples

AbstractThe comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.

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Detection of Oxidised Purines and UV-induced Photoproducts in DNA of Single Cells, by Inclusion of Lesion-specific Enzymes in the Comet Assay

Alternatives to Laboratory Animals ◽

10.1177/026119299602400315 ◽

1996 ◽

Vol 24 (3) ◽

pp. 405-411 ◽

Cited By ~ 17

Author(s):

Mária Dušinská ◽

Andrew Collins

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Single Cells ◽

Dna Strand Breaks ◽

Human Populations ◽

Dna Damage And Repair ◽

Strand Breaks ◽

Dna Lesion ◽

Dna Strand ◽

Uv Photoproducts

The comet assay (single cell gel electrophoresis) is a rapid, very sensitive method for the detection of DNA strand breaks at the level of single cells, which is now being applied in genotojricity testing. We modified this method for the detection of a variety of kinds of DNA lesion, by treating nucleoid DNA in the gel with either formamidopyrimidine-DNA glycosylase (which recognises ring opened purines, 8-hydroxyguanine and apurinic/apyrimidinic sites), or uvrABC excinuclease (uvrABC; which has a rather broad specificity, including bulky lesions and UV photoproducts). By using this modified assay, we demonstrate the removal of DNA strand breaks and oxidised purines upon incubating cells after treatment with hydrogen peroxide. This modification clearly increases the usefulness of the assay for the analysis of DNA damage and repair, for screening human populations for DNA damage, and for testing novel chemicals for genotoxicity.

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Collection and storage of human white blood cells for analysis of DNA damage and repair activity using the comet assay in molecular epidemiology studies

Mutagenesis ◽

10.1093/mutage/geab012 ◽

2021 ◽

Author(s):

Peter Møller ◽

Ezgi Eylül Bankoglu ◽

Helga Stopper ◽

Lisa Giovannelli ◽

Carina Ladeira ◽

...

Keyword(s):

Dna Damage ◽

Comet Assay ◽

Molecular Epidemiology ◽

Blood Cells ◽

Mononuclear Cells ◽

White Blood Cells ◽

Minor Effect ◽

Dna Damage And Repair ◽

Repair Activity ◽

Epidemiology Studies

Abstract DNA damage and repair activity are often assessed in blood s#38les from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the s#38les on the day of collection without any type of storage. For instance, certain studies use repeated s#38ling of cells from the same subject or s#38les from different subjects collected at different time-points, and it is desirable to analyse all these s#38les in the same comet assay experiment. In addition, flawless comet assay analyses on frozen s#38les opens up for the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors’ experiences indicate that various types of blood s#38les can be cryopreserved with only minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen s#38les of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB s#38les.

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