Comet Assay for DNA Damage Detection in Cells

Exposure to harmful agents induces single- and double-stranded breaks in the cellular DNA, which lead to DNA damage.

To detect DNA damage, begin with a single-cell suspension of chemical-treated cells in liquefied, low-melting point agarose. Load the suspension onto a glass slide, and allow the agarose to solidify — enabling the immobilization of cells within the porous gel matrix.

Treat the cells with lysis buffer. The detergents in the buffer solubilize the cellular and nuclear membranes, while the salts remove the histones from the DNA, forming a nucleoid body containing DNA attached to the nuclear matrix.

Incubate the slides in an alkaline electrophoresis buffer. This causes the DNA to unwind and generate single-stranded fragments. Place the slide in an electrophoresis apparatus, and connect it to the power supply. This allows the migration of negatively charged DNA toward the positive anode.

The undamaged DNA remains restricted within the nucleoid body. In contrast, the smaller, damaged DNA fragments begin moving toward the anode. This differential movement forms a comet-like structure, in which the undamaged DNA forms the head, while the damaged DNA forms the comet's tail.

Incubate the slide in a neutralization buffer to allow the single-stranded DNA fragments to renature. Next, treat the slide with a fluorescent dye that stains DNA. Visualize the slide under a fluorescence microscope.

The intensity and length of the tail correlate with the degree of cellular DNA damage.

To begin, prepare 0.6% low melting point agarose in PBS, and place it in a water bath at 37 degrees Celsius. Labeled the frosted end of the pre-coated slides with name, date, and treatment information using a permanent marker or pencil.

Place a chilling plate on a flat bench, and insert two frozen cooling packs into the sliding drawer below the metal surface. Then, place the slides on the chilling plate to pre-chill for 1 to 2 minutes. Centrifuge and remove the supernatant from the sample tubes, and place them immediately back on ice. Resuspend the cell pellet with 200 microliters of 0.6% low melting point agarose, and mix by pipetting.

Next, add 80 microliters of agarose-containing cells onto a chilled slide, and quickly place a coverslip on the gel. Allow the gel to set on the chilling plate for 1 to 2 minutes. In the meantime, prepare 500 milliliters of working solution of lysis buffer, and pour it into the lysis dish.

After the gels have set, remove the coverslips quickly by gently holding the slide between thumb and forefinger, and sliding the coverslip off the gel. Then, place the slides inside a slide carrier, with all the black dot marks on the slides facing in the same direction. Place the slide carrier inside the lysis dish. Close the lid of the lysis dish, and put it for incubation.

Carefully remove the slide carrier from the lysis dish without disturbing the gels. Gently, place the slide carrier in a washing dish pre-loaded with ice-cold double-distilled water. Ensure that the slides are completely submerged, and leave the setup for 30 minutes.

Place a frozen cooling pack under the electrophoresis tank to maintain optimal buffer temperature. Then, add ice-cold electrophoresis working solution to the tank, and transfer the slide carrier into it. Orient the slides, such that the cell containing gels point toward the cathode.

Incubate the slides in the electrophoresis tank for 20 minutes, to allow the DNA to unwind. Then, turn on the power supply to perform electrophoresis for 20 minutes at 1.19 volts per centimeter. After the electrophoresis is complete, turn off the power supply, and remove the slide carrier from the electrophoresis tank. Allow the slide carrier to drain on tissue paper for 30 seconds.

Next, place the slide carrier into a dish containing neutralization buffer, and leave it for 20 minutes. Following this, place it in a washing dish containing ice-cold double-distilled water, and leave it for 20 minutes. After washing, remove the slide carrier from the water, and allow the slides to dry in an incubator at 37 degrees Celsius for 1 hour.

To rehydrate the slides, transfer the slide carrier to a washing dish containing ice-cold double-distilled water and leave it for 30 minutes. Then, place the slide carrier into a staining dish containing 2.5 micrograms per milliliter propidium iodide solution.

Close the lid of the staining dish, and incubate it for 20 minutes in the dark at room temperature. After incubation, transfer the slide carrier to a separate dish, and wash it with ice-cold double-distilled water for 20 minutes.

Remove the slide carrier from the dish, and dry it completely in the dark, either in a 37 degrees Celsius incubator or at room temperature. After the slides dry completely, store them in a slide box in the dark until ready for image analysis.