Comet Assay for DNA Damage Detection in Cells

Abstract

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).

The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Protocol

1. Preparation of materials for the comet assay Preparation of the microscope slides Pour 1% (w/v) normal melting point agarose [dissolved in double-distilled water (ddH2O)] in a 50 mL tube and microwave to dissolve the agarose in the ddH2O. Store at 37 °C to prevent solidification prior to coating slides. Should solidification occur, discard and prepare fresh. Pre-coat microscope slides by dipping the slides into the 50 mL tube containing 1% (w/v) normal melt…

Representative Results

Figure 1: Representative images of a comet assay slide and HTP rack (microscope slide carrier). (A) For correct orientation, the pre-coated face of the microscope slide is recognized by a black dot in the right-hand corner of a microscope slide. (B) The image of the HTP rack illustrates how the slides are kept in a tight vertical orientation, with tabs on the carrier to fix its orientation within…

Materials

22 x 22 mm glass coverslips	Fisher Scientific, Hampton, NH, USA	631-0124
Fluorescence microscope equipped  with a camera	Zeiss, Jena, Germany
HTP Comet Assay system	Cleaver Scientific	COMPAC- 50
Human Keratinocyte (HaCaTs)	American Type Culture Collection  (ATCC), Manassas, VA, USA	Discontinued	Can be purchased from another  company  ADDEXBIO TECHNOLOGIES  Cat# T0020001
Hydrogen peroxide (H2O2)  30% in water	Fisher Scientific, Hampton, NH, USA	BP2633-500
Image and Data Analysis software	Perceptive Instrument,  Bury St Edmunds, England,  UK	125525	Free image analysis softwared is  available e.g., ImageJ
Low melting point Agarose	Invitrogen  Waltham, MA, USA	P4864
Na2 EDTA (disodium  ethylenediaminetetraacetic acid)	Sigma Aldrich,  St. Louis, MO,  USA	E5134
NaCl (Sodium chloride)	Sigma Aldrich,  St. Louis, MO,  USA	S7653
Nanopure Infinity Ultrapure Water  System (Barnstead Nanopure)	Thermo Scientific,  Waltham, MA,  USA	D11901	Ultrapure water (16 MΩ cm^{-1} )
NaOH (sodium Hydroxide)	Sigma Aldrich,  St. Louis, MO, USA	E5134
Normal melting point Agarose	Fisher Scientific,  Hampton, NH, USA	16520100	For pre-coating slides
Propidium Iodide  (1.0 mg/mL in water)	Sigma Aldrich,  St. Louis, MO,  USA	12-541BP486410ML
Single-frosted glass microscope  slides	Fisher Scientific,  Hampton, NH, USA	12-541B
Slide box	Fisher Scientific,  Hampton, NH, USA	03-448-2	Light proof, to protect cells from  the formation adventitious damage  (according to the widely held view)  and prevent fading of the fluorescent  dye
Slide Chilling plate	Cleaver Scientific,  Rugby, England,  UK	CSL-CHILLPLATE
Treatment dish	Cleaver Scientific,  Rugby, England,  UK	STAINDISH4X
Tris-base	Sigma Aldrich,  St. Louis, MO,  USA	93362
Triton X-100	Fisher Scientific,  Hampton, NH, USA	BP151-500
Trypsin EDTA (0.5%)	Invitrogen  Gibco,  Waltham, MA, USA	15400054
Vertical Slide Carrier	Cleaver Scientific,  Rugby, England,  UK	COMPAC-25