DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation
Trascrizione
Plate 100,000 cells in triplicate wells in a 24-well plate. After 46 hours, add tritiated thymidine to the culture medium and incubate at 37 degrees Celsius for 2 hours.
Then, after removing the medium, add 300 microliters of pre-warmed 0.25% trypsin-EDTA and incubate for 20 minutes at 37 degrees Celsius to break the cells apart and release the DNA. Then, turn the cell harvester and pump on.
Place the filter paper in the cell harvester. Keep collecting the tubes of the cell harvester in an empty blank tray. Then, press pre-wash to wet the filter paper. Place the collecting tubes in sample wells and start.
Use a hot light source to dry the filter paper and arrange corresponding vials on the tray. Punch out paper chads into the vials and then add 2 milliliters of liquid scintillation cocktail to each vial.
Incubate the vials for 1 hour in the scintillation counter before operating the machine to obtain the counts per minute of each sample, which reflects the active DNA synthesis.
