A novel triple label method validates the double label technique of defining cell cycle kinetics in HL-60 cells.
HL-60 cells were sequentially labeled with the thymidine analogues iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). The labeling index (LI), the duration of S-phase (Ts) and the total cell cycle time (Tc) were measured immediately. It was therefore possible to predict the next time when the single versus double labeled cells would re-enter the S-phase. In our study, the Tc was calculated to be 20 hours. The third label, tritiated thymidine (3HTdR), was introduced at the predicted time of 20 hours to confirm the validity of the previously calculated Tc. The actual percentage of cells which were labeled by (3HTdR) was very similar to the predicted value. We conclude, therefore, that the calculated cell cycle time correlated well with the actual cell cycle time, at least in a controlled in vitro culture system. This novel triple label method validates our double-label technique developed for cell cycle measurements.