The overall goal of this experiment is to investigate the effectiveness of genotoxic agents in cancer cells using a single cell electrophoresis assay, which is also called the comet assay. This procedure is a sense to measure the four genetic toxicological studies. And it is applicable to different evals, such as earlier drug candidate selection and the fundamental research in DNA damage and the repair.
This technique provides a direct way to quantitatively evaluate the DNA damage. And, it is efficient, easy to perform, and relatively inexpensive. Prepare the agarose for slide coating by melting one percent agarose in a microwave for two to three minutes or until the agarose is completely molten.
Dip a glass microscope slide into the agarose and use a lint-free wipe to wipe one side of the slide. Lay the slide on a flat surface to air dry. A transparent agarose film should be formed after drying.
Place the coated slides in 37 degrees Celsius before use. The therapeutic effect of chemotherapy is evaluated in U251 glioma cells cultured in DMEM/Ham F-12 medium supplemented with 10%FBS, 100 units per milliliter of penicillin and 10 micrograms per milliliter of streptomycin. Digest the cells using one milliliter of trypsin for three minutes, and then neutralize the trypsin using DMEM/Ham F-12 medium with FBS.
Collect the cells in a 15 milliliter tube. And spin at 300 times G for four minutes. Aspirate the medium.
And suspend the cells at two times 10 to the fifth cells per milliliter in 1x PBS. Cells and should be prepared immediately before starting the assay. And the should be handled in the dark.
All in dim light to prevent damage. Combine the cell suspension with one percent molten, low melting point agarose at a volume ratio of one to 10. Mix gently by pipetting up and down.
And immediately pipette 30 microliters on to a slide. Use the slide of the pipette tip to spread the agarose mixture to ensure the formation of a thin layer. Place the slides flat at four degrees Celsius, in the dark, for 10 minutes.
Immerse the slides in four degree Celsius lysis solution, in the dark for one hour to overnight. For the alkaline comet assay, gently remove slides from the lysis solution, drain excess liquid, and gently immerse slides in AES, in the dark at four degrees Celsius for one hour to allow DNA unwinding. Add pre-chilled AES in the electrophoresis slide tray.
Do not exceed 0.5 centimeters above the slides. Place the slides inside and cover with a cap. Set the power supply voltage to one volt per centimeter and run at four degrees Celsius for 30 minutes.
When the electrophoresis is complete, drain excess AES from the slides. Gently immerse the slides twice in distilled water at room temperature for five minutes each time. After that, gently immerse the slides in 70%ethanol at room temperature for five minutes.
For the neutral comet assay, gently remove the slides from the lysis solution, drain excess liquid, and gently immerse in NES in the dark, at four degrees Celsius for 30 minutes. Add pre-chilled NES in the electrophoresis slide tray, not exceeding 0.5 centimeters above the slides. Place the slides inside and cover with a cap.
Set the power supply voltage to one volt per centimeter and run at four degrees Celsius for 45 minutes. Drain excess buffer from the slides. Gently immerse the slides in DNA precipitation solution at room temperature for 30 minutes.
After 30 minutes, gently immerse the slides in 70%ethanol at room temperate for 30 minutes. Begin this procedure by drying the slides in the dark at 37 degrees Celsius for 10-15 minutes. Next, place 50-100 microliters of green fluorescent nucleic acid staining solution on to the dried agarose of each slide, and stain for 15 minutes at room temperature in the dark.
Rinse the slides briefly in distilled water, and dry completely at 37 degrees Celsius in the dark. Perform image acquisition and analysis immediately after staining the comet slides. Place a slide under a fluorescent microscope with a slide holder.
Select the 10X objective lens, and ensure the agarose gel is facing the objective lens. Randomly capture images from each stained comet slide. Avoid the edges and the areas around any air bubbles.
Ensure each comet tail is horizontally distributed. Comet heads should originate from the left, and the tail from the right. Save each picture in a binary tive format with bright DNA stain and dark background.
For image analysis, first load all images to the comet assay software, using the Select Files to Analyze button, which is located on the left of the tool bar. An image view window should appear. Draw a measurement frame on the screen, and adjust its size with accordance with the comet of the cell.
Click adjust to set up the threshold of the head, comet and tail, according to the image. And then click Start Measurements. Select a cell using the frame and activate the measurement by clicking on the Assay the Comet button.
An intensity image will show up on the profiles window, with the selected measurement parameters. Save the results by clicking the Store Results button. Analyze at least 50 cells per treatment.
Results from the Alkaline and Neutral comet assays show that the comet tails of doxorubicin treated UT51 cells were longer and had higher DNA intensity, suggesting a substantial accumulation of fragmented DNA. Quantitative analysis for either the Alkaline or Neutral comet assay, showed significantly increased comet tail formation, after doxorubicin treatments, indicating DNA damage. A combination treatment of a PARP inhibitor, olaporib, and an alkylating agent, temozolomide, significantly enhanced DNA damage in glioma cells, reflected by the increased length and intensity of the comet tail signal.
Olaporib alone, did not introduce DNA damage. Where as it potentiated the genotoxic effect of temozolomide. Representative images of glioma cells under single and combination treatments, showed the longer and higher intensity comet tails of cells treated with temozolomide and olaporib.
Once mastered, this technique can be done in five hours, if it is performed properly. After its development, this technique paved the way for researchers in the field of genotoxicity to measure DNA string breaks in many types of ukaryotic cells. Such as cultured cells or cells isolated from organisms.
After watching this video, you should have a good understanding of how to use the comet assay to investigate the effectiveness of a genotoxic agent at the single cell level. Don't forget that working with the damage reagent can be extremely hazardous. And the precautions, such as wearing a lab coat and gloves, should always be taken while preforming this procedure.
