Measuring DNA modifications with the comet assay: a compendium of protocols

Abstract

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/ apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Impact of Deoxynivalenol and Zearalenone as Single and Combined Treatment on DNA, Cell Cycle and Cell Proliferation in HepG2 Cells

“…MACSQuant ® Analyzer 10 (Miltenyi Biotech, Bergisch Gladbach, Germany) flow cytometer using FlowJo v10 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used for the analysis. FITC intensity correlating to DSBs was detected in the B1-A channel, PE Vio770 intensity correlating to KI67 positive cells was detected in the B3-A channel and Hoechst fluorescence correlating to cell cycle was detected in the V1-A channel as reported by Hercog et al [ 48 ] and Ujvarosi et al [ 51 ]. To exclude unspecific binding, we used internal REA controls.…”

“…The alkaline comet assay was performed according to Collins et al [ 48 ] following recommendations by Møller et al [ 49 ]. HepG2 cells at density 80,000 cells/well were seeded onto 12-well plates.…”

“…In experiments, negative control (cell medium), solvent control (0.1% DMSO) and positive control (1 μg/mL ET) were included. Cells were first fixed in 70% ethanol, washed in cold PBS and then labelled with anti-KI67-PE Vio770 and anti-H2AX pS139-FITC (diluted 1:50 in 1% BSA) for 30 min, washed again with PBS, and afterwards incubated with Hoechst 33258 dye (diluted 1:500 in 0.1% Triton X-100) for 20 min as reported by Hercog et al [ 48 ] and Štampar et al [ 50 ]. MACSQuant ® Analyzer 10 (Miltenyi Biotech, Bergisch Gladbach, Germany) flow cytometer using FlowJo v10 software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used for the analysis.…”

Particle Debris Generated from Passenger Tires Induces Morphological and Gene Expression Alterations in the Macrophages Cell Line RAW 264.7

“…The alkaline comet assay was performed according to Singh et al [ 42 ] and Collins et al [ 43 ] using Trevigen’s comet slide (2 wells).…”

Combined Approach: FFQ, DII, Anthropometric, Biochemical and DNA Damage Parameters in Obese with BMI ≥ 35 kg m−2

“…For the alkaline and Fpg comet assays, 2 mL of whole fresh human blood from each volunteer was collected in an EDTA vacutainer, transported to the Institute for Medical Research and Occupational Health at 4 °C, light protected, on the same day, and immediately processed further. The procedure for the alkaline comet assay was described previously [ 34 ], and details can also be seen in the compendium of protocols (Nature protocols) [ 35 ], but we will repeat it here again. Comet slides were already prelayered with 300 μL of 1% normal melting point (NMP) agarose and dried.…”