The comet assay is widely used for assessing genotoxicity. Our protocol offers a number of methodological advantages over the standard comet assay. Our technology centers on holding the standard comet slides vertically and being processed in batches of 25 in a purpose-made rack.
This includes electrophoresis, so the tank is smaller, uses less buffer, and has integrated cooling. Being held in a rack, the delicate gels on the slides are more protected and move from solution to solution in 25 seconds. Using our blood comet protocol, researchers can use a pin prick of blood to study DNA damage in humans, other species, and a wide variety of potentially genotoxic environmental exposures.
Any of the different formats for the comet assay that use microscope slides as the solid support for the comet gels are amenable to our approach. The crucial part of this technique is loading samples after mixing with LMP agarose. Samples should be loaded quickly without making bubbles and covered with a cover slip right away before solidifying.
To begin, prepare 0.6%low-melting point agarose in PBS and place it in a water bath at 37 degrees Celsius. Label 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 one to two 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 cover slip on the gel. Allow the gel to set on the chilling plate for one to two 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 cover slips quickly by gently holding the slide between thumb and forefinger and sliding the cover slip 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 preloaded 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 one 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. Human keratinocytes were irradiated with different doses of UVA and UVB or treated with 50-micromolar hydrogen peroxide to induce DNA damage.
The optimal voltage was 1.19 volts centimeter, as it induces the most sensitive response and the greatest percentage of tail DNA. Human whole blood was irradiated with different doses of UVA and treated with different concentrations of Fpg. High-throughput alkaline comet assay revealed that the optimal levels of DNA damage were induced with four units per milliliters of Fpg.
The ovarian cancer cell line SK-OV-3 was treated with a combination of cisplatin and hydrogen peroxide. The unexposed cells showed no damage. Exposure to hydrogen peroxide alone generated a significant mean Olive tail moment compared to the cells in which DNA intrastrand crosslinks were induced.
After treatment with 100-micromolar cisplatin, DNA intrastrand crosslinks increased with a peak at 12 hours, after which the levels decreased to zero after 30 hours It is essential to put the ice pack in the electrophoresis tank and incubate slides for 20 minutes to unwind DNA before the electrophoresis. This step will help the damaged DNA to travel by current. Running the assay with slides placed vertically allowed us to automate the comet assay.
Additionally, our technique has simplified the comet assay for research.
