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 |
