Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Summary

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

Abstract

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.

The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell ^1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

Protocol

1. Cell Culture

1. Culture neuronal cells and treat them as needed.
2. Harvest cells into 15 ml tubes: aspirate media, rinse with phosphate buffered saline (PBS, calcium and magnesium free), add trypsin, collect in 15 ml tubes and neutralize trypsin with appropriate serum containing media.
3. Spin at 1,000 x g for 5 min.
4. Aspirate media.
5. Resuspend cells in PBS.
6. Spin at 1,000 x g for 5 min.
7. Aspirate media and resuspend cells in fresh PBS.
8. Count cells using a hemacytometer or your preferred cell counter.
9. Cell samples should be prepared immediately before starting the assa....

Discussion

The comet assay has the unique capacity of analyzing individual cells. This is advantageous in identification of subpopulations of cells that demonstrate differential response to cytotoxic agents. A few practical limitations have to be taken into account. The number of cells that can be evaluated individually may vary depending on the individual. The sample size needs to be increased if there is variance in DNA damage within a population. Viable single-cell suspension is critical for this assay since predominant presence.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 ml tubes	Santa Cruz Biotechnology, Inc	Sc-200271
10X TBE (Tris base, boric acid, EDTA)	Fisher Scientific	BP13331
15 ml tube	Fisher Scientific	0553851
Agarose	Sigma	A5093
Aluminum foil	Fisher Scientific	01213101
Beaker	Fisher Scientific	FB102300
Centrifuge	Thermo Scientific	75004261PR
Comet Assay software	Comet Assay IV Image Analysis System
Cylinder	Fisher Scientific	08555F
EDTA	GIBCO	15575
Electrophoresis chamber	Thermo Scientific	09528101
Ethanol	Fisher Scientific	A407P4
Fluorescent microscope	Zeiss Axio Vision		Any fluorescent microscope with green filter will suffice
Hemacytometer	Fisher Scientific	0267152
Low melting point agarose	Promega	V2111
Microwave	Sears
Phosphate buffered saline, calcium free, magnesium free	HyClone	SH3025601
Power supply	BioRad	1645050
Refrigerator	Sears
Ruler	Staples
Slide	Fisher Scientific	12550143
Sodium chloride	Sigma	S7653
Sodium hydroxide	Sigma	S5881
Sodium lauryl sarcosinate	Fisher Scientific	S529
Sybr green	Invitrogen	S7585
Tray	Fisher Scientific	15242B
Tris Base	Sigma	T6066
Triton-X 100	Sigma	T8787
Trypsin	HyClone	SH3023601
Vortex	Fisher Scientific	02216108
Water bath	Fisher Scientific	154622Q