Comet Assay for DNA Damage Detection in Cells

Published: April 30, 2023

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…

開示

The authors have nothing to disclose.

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

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記事を引用
Comet Assay for DNA Damage Detection in Cells. J. Vis. Exp. (Pending Publication), e21317, doi: (2023).

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