Neutral comet assay was performed as previously described (40 (link)). Briefly, 700 cells were resuspended in 70 μl 0.5% low melting point agarose (Trevigen, Cat. No. 4250-050-02) and spread on a comet slide (Trevigen, Cat. No. 4250-200-03). Cells were lysed in a cold lysis solution (Trevigen, Cat. No. 4250-050-01) at 4°C for 30 min. DNA migration was performed in TBE buffer at 1 V/cm for 30 min. Slides were washed in milliQ water, fixed with ethanol 70% for 30 min and dried at room temperature. Comets were labeled with SYBR® Gold Nucleic Acid Gel Stain (ThermoFisher) for 30 min. Images were acquired with a fluorescence microscope (LEICA DMU 4000B; 20×/0.4 CORR) coupled to the LEICA DFC345FX camera. The images were analyzed using the OpenComet plug-in from ImageJ. At least 100 comets were scored per sample in each experiment. Olive moment measures DSBs as a product of the amount of DNA present (intensity) times the size of the broken DNA pieces (length) in the tail.
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Neutral Comet Assay for DNA Damage
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Corresponding Organization : Saint Louis University
Other organizations : Washington University in St. Louis, Vanderbilt University, Université Laval
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Variable analysis
independent variables
- None explicitly mentioned
- Olive moment, which measures DNA double-strand breaks (DSBs) as a product of the amount of DNA present (intensity) times the size of the broken DNA pieces (length) in the tail
- Number of cells (700 cells resuspended)
- Agarose concentration (0.5% low melting point agarose)
- Lysis conditions (cold lysis solution at 4°C for 30 min)
- DNA migration conditions (1 V/cm for 30 min in TBE buffer)
- Slide preparation (spread on comet slide, washed in milliQ water, fixed with 70% ethanol for 30 min and dried at room temperature)
- Staining (SYBR® Gold Nucleic Acid Gel Stain for 30 min)
- Imaging conditions (fluorescence microscope with 20×/0.4 CORR objective, LEICA DMU 4000B coupled to LEICA DFC345FX camera)
- None explicitly mentioned
- None explicitly mentioned
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