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Toxys is a Dutch biotech company that provides innovative in vitro toxicity screening solutions to rapidly identify hazardous and potential carcinogenic properties of novel compounds during the early phases of product and drug development.

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In vitro comet assay

The comet assay can be used to detect DNA damage. We perform the assay using our ToxTracker cells, to add another endpoint/readout to the assay.

Key Features

  • DNA damage assay
  • Performed with the ToxTracker cells
  • Detects double strand breaks and single strand breaks

Comet assay, in alkaline and neutral 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 version offers increased sensitivity when investigating agents that cause DNA strand breaks or induce DNA lesions that are alkali-labile. In fact, 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 the gel has solidified, the slides are placed in a lysis solution. This solution contains Triton X-100, which will break down membranes and a high concentration of salt which will remove histones and other soluble proteins. When the slides are then incubated in alkaline electrophoresis buffer, DNA will unwind and thus single-stranded DNA is obtained. Electrophoresis is then performed under alkaline conditions. The electric current pulls DNA from the nucleus toward the anode, which results in an image that looks like a comet with a head and a tail (Figure 1).

The more strand breaks that are present in DNA, the more DNA will be present in the tail. After neutralization, the slides fixed DNA is stained with a DNA-specific dye, e.g., ethidium bromide, propidium iodine, or SYBR Green, and the comets are examined with a microscope. From the microscopic images, the amount of tailing is determined, typically expressed as % DNA in the tail.

Applications

Typically we perform the in vitro comet assay as add-on to the ToxTracker assay. We can perform the assay in both neutral and alkaline conditions and use the ToxTracker cells for the assay.

Image based data

For this assay we will collect images. Primary Comet data are generated as .bmp or .jpg microscopy images. Automated image analysis using the CometScore plugin for ImageJ provides .txt files with comet quantification data. All test results are collected, summarised and supplied to customer in a Microsoft Excel file. The amount of tailing is determined and typically expressed as % DNA in 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 by the tested compounds. Fold changes represent increase in %DNA in the tail after 0.5 or 4 h. exposure, compared to untreated cells.

Fee for Service Project

We can perform the assay for you at our state-of-the-art laboratory. We are experience in tailoring assay to meet your demand, so please contact us if you are interested in the additional assay we can run in parallel with the ToxTracker assay.

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