Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy
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Combine the cell suspension with 1% molten low melting point agarose, at a volume ratio of 1:10. Mix gently by pipetting up and down, and immediately pipette 30 microliters onto a slide. Use the side of the pipette tip to spread the agarose-cell mixture to ensure the formation of a thin layer. Place the slides flat at 4 degrees Celsius in the dark for 10 minutes. Immerse the slides in 4 degrees 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 4 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 1 volt per centimeter, and run at 4 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.
Begin staining procedure by drying the slides in the dark at 37 degrees Celsius for 10 to 15 minutes. Next, place 50 to 100 microliters of green fluorescent nucleic acid staining solution onto 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.
