In vitro comet assay

Comet assay, in alkaline conditions

The Single-cell gel electrophoresis (comet assay) was first developed in 1984 by Östling and Johanson to analyse DNA strand breaks under neutral conditions. Some years later, Singh et al. introduced an alkaline version (pH >13) of the method that enables the detection of single-stranded DNA breaks (SSB). The alkaline condition of the Comet assay offers increased sensitivity when investigating agents that cause DNA strand breaks or induce DNA lesions that are alkali-labile. The alkaline comet assay is now often the method of choice if low levels of DNA damage are to be detected, e.g., in lymphocyte samples from the human population or in in vitro and in vivo testing of genotoxicity.

In the comet assay, cells are embedded in low-melting-point agarose gel on a microscope slide. After lysis of the cells, slides are incubated in alkaline electrophoresis buffer and DNA fragments that are caused by DNA strand breaks are separated by electrophoresis. DNA-specific dyes, e.g., ethidium bromide, propidium iodide, or SYBR Gold are used to visualise the DNA comets, as shown in the figure below. The percentage of DNA in the tail is quantified as the measure for the induction of DNA strand breaks.

FPG-modified comet assay

To specifically detect oxidative DNA lesions, the microscope slides can be treated with formamidopyrimidine DNA glycosylase (FPG). The FPG enzyme cleaves purines that have been damaged by oxidation, such as 8-oxo-7,8-dihydroguanine (8-oxoG) lesions, leaving a single strand break. In the alkaline comet assay, this damaged DNA then appears in the tail. By comparing the DNA in the tail in the absence and presence of the FPG enzyme, the extent of oxidative damage can be assessed.

Applications

Typically, we perform the in vitro comet assay as an add-on to the ToxTracker assay. The comet assay is also performed in the various DNA repair-deficient cell lines that are applied in the DNA Repair-Profiler assay.

Image-based data

For the comet assay, we will collect microscope images of the DNA comets. Automated image analysis is performed using the CometScore plugin for ImageJ. All test results are collected, summarised and included in a report that will be shared with the customer. The amount of DNA strand breaks is determined and typically expressed as % DNA in the tail. The induction levels of comet tails by test compounds are subsequently expressed as fold changes compared to untreated cells.

Example result: Induction levels of comet tails in the absence and presence of the FPG-enzyme by the tested compounds after 4h exposure. The graph below shows the %DNA in the tail in the absence and presence of FPG-enzyme.

The below graph shows that the induction of FPG-sensitive DNA adducts by potassium bromate (KBrO3) can be detected in the modified comet assay.

Service

We perform the in vitro comet assay as a service for our clients. You can send your compounds and receive a full report within 3-4 weeks. Are you interested in receiving a quote or do you have any questions? Please reach out!

Practical information

The amount of compound that is required for analysis in the in vitro comet assay depends on the maximum concentration that will be applied in the project.

Turn around time

• 3-4 weeks

Type of solvents compatible with the assay

• DMSO
• PBS
• Water
• Other solvents can be discussed with our Study Director

A standard in vitro comet assay report includes

• Dose-range finding analysis (done with Alamar Blue)
• Graphical representation of mean tail % and fold change (-FPG enzyme vs +FPG enzyme)

Next steps after sharing your interest in the in vitro comet assay