The overall goal of this procedure is to measure and quantify the amount of DNA damage in neurons using the COMET assay. This is accomplished by first exposing the neurons to various DNA damaging conditions, collecting the neurons, and mixing them with low melting point aros. The second step is to immobilize the neurons on slides.
Next, the neurons are lysed and then subjected to electrophoresis in the final step. The DNA is stained with cyber green. Ultimately fluorescence microscopy and the COMET assay software are used to visualize and quantify the levels of DNA damage in individual neurons.
Hello, my name is Eddie Yang and I am an assistant professor in the Department of Radiation Oncology at the University of Alabama at Birmingham. Today, we will be demonstrating the COMET assay, which is a simple method to detect DNA damage in cells. The main advantage of this technique over other existing methods such as the Gamma H two A X foci staining, as well as the pulse field gel electrophoresis, is that this technique is a direct and sensitive method to measure DNA damage.
Additionally, with minor modifications of this technique, the type of DNA damage can be determined because many cancer therapies act by inducing DNA damage in tumors and often act by altering DNA repair capacity. The implications of this technique can be extended toward the efficacy of cancer therapy. Demonstrating the procedure today will be Ms.Samara Noshin, a research assistant from my laboratory, Begin by harvesting neuronal cells treated under the desired experimental circumstances into 15 milliliter tubes.
Spin down the tubes for five minutes at 1000 times G and four degrees Celsius after aspirating the supernatant resus. Suspend the pellet in PBS and then spin down the cells again after the second wash. Resus suspend the cells in more PBS, count them and keep them in the dark until used in the comet assay.
To avoid DNA damage before beginning the come assay melt 1%aros in a microwave for three minutes until all the granules disappear. Then dip the slides into the molten agros and wipe one side clean with a Kim wipe. Allow the agros to air dry to a transparent film while the agros is drying chill lysis solution at four degrees Celsius for at least one hour and melt more.
1%low melting point agros.Cool. This batch of agros to 37 degrees Celsius in a water bath for half an hour. To avoid the artificial induction of a comet tail, now dilute the harvested neuronal cell suspension so that there are 100, 000 cells per milliliter.
Combine the cell suspension with the 37 degrees Celsius low melting point agros at a one to 10 ratio vortex briefly and immediately use the side of a pipette tip to spread 50 microliters of the cell suspension evenly over the sample area of the air dried comet Slide. Place the treated slides flat in the refrigerator for 30 minutes until a circle appears in the periphery of the slide. Then submerge the slides in the pre chilled lysis solution in the dark at four degrees Celsius.
After 30 minutes, aspirate the lysis solution. Then incubate the slides in one x neutral electrophoresis buffer for half an hour in the refrigerator. Next, add pre chilled one x neutral electrophoresis buffer to an electrophoresis chamber, and then place the slides in the electrophoresis slide tray, aligning the slides so that they're equally distant from the electrodes.
After filling the chamber enough to cover the slides with 0.2 inches of one x neutral electrophoresis buffer, set the power supply voltage to one volt per centimeter and run the assay in the dark for 30 minutes at four degrees Celsius. Begin by immersing new agros and neuronal cell treated slides in fresh pre chilled lysis solution, and then incubate them in the dark at four degrees Celsius for one hour to overnight. Next, remove the slides from the lysis solution, drain them, and then rinse them once with cold neutralization buffer for five minutes to remove any residue prior to the alkaline winding step.
Then place the slides in a gel electrophoresis chamber filled with pre chilled, freshly made electrophoresis buffer not to exceed 0.5 centimeters above the slides. Incubate the slides in the alkaline buffer for 30 minutes in the dark to allow for the unwinding of the DNA and the expression of the alkali liable damage. Then run the assay at one volt per centimeter for 30 minutes at four degrees Celsius.
After running the comet assay, drain the excess electrophoresis buffer and then immerse the slides in pre chilled distilled water at room temperature. After five minutes, immerse the slides in pre chilled, 70%ethanol also at room temperature. After five more minutes, set the samples to dry overnight, keeping them out of bright light.
Then stain the slides in cyber green in the refrigerator for 30 minutes. Now remove the slides and allow them to dry completely in the dark. The aros will become transparent when completely dry.
To acquire images from the assay, use a fluorescent microscope set to the green filter. Then open the COMET assay program to analyze the acquired images. By clicking on the comet head button, the software will calculate the parameters including the mean tail moment and the amount of DNA in the nucleus.
After analyzing at least 200 cells per treatment, export the data to Microsoft Excel for calculation of the mean tail moment and data plotting. This first figure shows an example of a COMET assay analysis of neuronal cells with and without a comet tail. In this second comet assay analyzed using the COMET assay software, irradiation of the neuronal cells was determined to induce DNA damage as the cells were subjected to the electrical field.
The DNA migrated at different rates due to differences in size as subsequently analyzed with the common assay software. This first screenshot shows representative images acquired using the fluorescent microscope using the COMET assay program. Clicking on the nucleus or the comet head of one image generated a fluorescent map graph and data table.
The more the damage to the DNA, the farther the DNA migrated out of the nucleus. This resulted in a decrease in the fluorescence intensity in the nucleus as indicated by the green color, and is subsequently detected by the software producing a higher tail moment. In this final figure, a representative graph of the mean tail moment obtained by analyzing the DNA damage in irradiated and non irradiated neuronal cells using a neutral COMET assay is shown as expected three gray radiation induced DNA damage as indicated by the higher mean tail moment in the irradiated neuronal cells.
While attempting this procedure, it is important to remember to handle the cells carefully preventing exposure to direct light and control the temperature in pH. This is so that the cells are not subjected to additional stress, which could alter your results Following this procedure. Other methods, such as analysis of DNA, repair foci via immunofluorescence staining can be performed in order to answer additional questions about which DNA repair pathways are altered.
For example, immunofluorescent staining for RAD 51 foci or phospho, DNA PK Foci can be performed to measure homologous recombination repair or non-homologous, and joining repair respectively.
