To begin, remove the 1.5 milliliter centrifuge tubes containing 100 microliters of 1%solidified argos from the four degrees Celsius storage. Place the tubes in a water bath at 42 degrees Celsius to melt the agros. Mark the sample identifications on the frosted end of the two well comet slides.
Seed adherent U2 OS cancer cells in a six well dish containing DMEM and culture overnight at 37 degrees Celsius with 5%carbon dioxide. The next day to demonstrate replication dependent break induction treat cells with one micromolar Camptothecin or CPT for one hour. After treatment aspirate the medium and rinse the cells twice with PBS, add 300 microliters of trypsin to each well.
Incubate the dish in a tissue culture incubator for two to three minutes. Now add 700 microliters of DMEM to each well. Resuspend gently and transfer the suspension to 1.5 milliliter centrifuge tubes.
Centrifuge the cell suspension at 2, 400 G for five minutes. Remove the supernatant and resuspend the pellet in one milliliter of cold PBS. After the final centrifugation, resuspend the cells in one milliliter of PBS at a cell density of two times 10 to the power of five cells per milliliter.
Transfer 10 microliters of the cell suspension to a 100 microliter aliquot of melted argos. Using the same pipette tip mixed gently and spread the mixture evenly into one well of the slide. Store the slides at four degrees Celsius in the dark for 15 to 20 minutes.
After solidification, transfer the slides to coplan jars containing lysis buffer for one hour, rinse the slides once with a cold TAE buffer. Then immerse the slides in a cold TAE buffer for 30 minutes. Pour approximately 850 milliliters of cold TAE buffer into the electrophoresis apparatus.
Place the slides on the electrophoresis tray in the proper orientation to allow for the migration of broken DNA. Gently place the slide tray overlay on top of the tray to cover the slides. Connect the cables and set the power to 21 volts.
Perform electrophoresis for 40 minutes. After electrophoresis remove the slides and immerse them in a DNA precipitation solution for 30 minutes. Transfer the slides to a 70%ethanol solution for 30 minutes.
Dry the slides in a hybridization oven at 45 degrees Celsius for 30 minutes in the dark. Next, add 100 microliters of cyber green solution onto the slides and incubate in the dark. After 30 minutes, tilt the slide and remove the excess dye solution by gently dabbing the edge of the well with a lint-free wipe.
Dry the slides in a drawer for 30 minutes. After acquiring images for at least 100 comets per sample, open the COMET score software and export images score. The comets that are individually distinguishable and fully contained in the field of view.
Break induction significantly increased in U2 OS cells treated with CPT and hydroxyurea or HU compared to DMSO treated controls. Lysis temperature had a minimal effect on tail migration in untreated U2 OS cells. Prolonged HU treatment in U2 OS cells led to an increase in break induction over time.
Increased CPT concentration resulted in a dose-dependent increase in break accumulation in U2 OS cells. Basal DNA break levels varied among hTERT-RPE-1, HEK293T, and U2 OS cells with increased breaks observed following CPT treatment. Break accumulation was observed in U2 OS cells treated with HU CPT and PARP inhibitor as compared to DMSO controls.
