DNA Synthesis Assay: A Technique to Assess DNA Synthesis in Proliferating Cells Using Radioactive Tritiated Thymidine Incorporation
Transcript
Begin by taking a multi-well plate containing adherent cells.
Treat these cells with a media containing tritiated thymidine – a radioactive version of thymidine - and incubate.
During incubation, actively replicating cells incorporate radioactive thymidine. This thymidine pairs opposite adenosine, generating radiolabeled DNA in proliferating cells.
Remove the spent media from wells. Add a lysis buffer to lyse the cells and release DNA into the suspension.
Prepare the cell harvester assembly by placing the collecting tubes of the harvester over the wells of the microplate.
Apply suction pressure to enable the aspiration of suspended DNA from the wells. This DNA deposits on the filter paper, forming specific spots.
Cut out the filter paper chads with DNA spots. Transfer these chads individually into scintillation vials.
Overlay the filter paper with a scintillation cocktail. Place the vial in a liquid scintillation counter.
Inside the counter, the labeled DNA emits energy by radioactive decay. This energy is absorbed by the components of the scintillation cocktail. Eventually, the cocktail components begin to transfer the pulses, which in turn are absorbed by the sensor.
The amount of radioactivity detected determines the extent of cell division.
