This article describes a method for measuring DNA synthesis in proliferating cells using tritiated thymidine. The process involves treating cells with radioactive thymidine, lysing them, and quantifying the incorporated radioactivity to assess cell division.
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.
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.
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