The comet assay was used to measure DNA damage (directly) and DNA repair (indirectly): 1 × 106 cells from the cell lines MDA-MB-231 and MCF10A were isolated and exposed to H2O2 (50 mM) (Sigma Aldrich, UK), cortisol and NE for 20 minutes. Cells were then either processed immediately or washed in PBS and incubated at 37 °C for a further 20 minutes to allow DNA repair. Cells were then mixed with 1.2% low melting-point agarose (Sigma Aldrich, UK) and pipetted onto slides previously coated with 0.6% ultrapure agarose (Invitrogen, UK). The gels were allowed to set at 4 °C and lysed in comet lysis buffer (Trevigen) before immersion in electrophoresis buffer (50 mM NaOH, 1 mM EDTA, 1% dimethyl sulfoxide (DMSO)) for 45 minutes. Electrophoresis was carried out at 25 V for 25 minutes and the slides were neutralised in 0.4 M Tris pH7. Cells were stained with ethidium bromide and the “comet tails” of 100 cells were scored blind (Nikon, UK) using a 0–4 scoring system based on the length of the tails. Scores are expressed as arbitrary units out of a maximum score of 400.
Comet lysis buffer
Manufactured by Bio-Techne
Comet lysis buffer is a laboratory reagent used in the comet assay, which is a technique for measuring DNA damage in individual cells. The buffer is designed to lyse cells and expose the DNA for subsequent analysis. It contains a combination of detergents, salts, and other compounds that facilitate the lysis process. The buffer is a necessary component in the comet assay protocol, but its specific function is to prepare the cellular samples for the assay.
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2 protocols using comet lysis buffer
1
Comet Assay for DNA Damage and Repair
2
Neutral Comet Assay Protocol
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Neutral comet assays were performed according to the Trevigen protocol. Cells were trypsinized and resuspended in PBS at a density of 3 × 105 cells/mL. Cells were mixed at a 1:10 v:v ratio with molten 1% low melting point agarose (Trevigen) at 37°C, and 50 µL of cell-agarose solution was dropped onto Flare slides (Trevigen). The agarose was allowed to solidify at 4°C, and slides were soaked in ice-cold Comet lysis buffer (Trevigen) and incubated at 4°C overnight. Slides were then incubated for 30 min at 4°C in cold neutral electrophoresis buffer (100 mM Tris, 300 mM sodium acetate at pH 9.0). Slides were carefully immersed in neutral electrophoresis buffer in a Thermo Scientific Owl EasyCast B1A mini gel electrophoresis system gel tank and electrophoresis was carried out at 15 V (1 V/cm) for 1 h at 4°C. Slides were sequentially incubated for 30 min in DNA precipitation solution (1 M ammonium sulfate, 95% ethanol) and 70% EtOH. Slides were dried and 200 µL of SYBR Gold DNA staining solution (1:10,000 SYBR Gold, 10 mM Tris at pH 7.5, 1 mM EDTA) was dropped onto each sample. Slides were incubated 30 min at room temperature, washed twice with H2O, and completely dried before mounting with ProLong Gold. Images were captured using NIS elements software with a Nikon i90 microscope and tail moments were analyzed with the OpenComet software integrated in Fiji (Gyori et al. 2014 (link)).
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