Allowing for endogenous dTTP

The problems with flooding the pool are the assumptions generally made that the concentration of thymidine used is (a) sufficient to flood the pool and (b) low enough not to have any deleterious effects on cell growth. 

The rate of incorporation of radioactivity is dependent on the specific activity of the [3H]dTTP which in the two previous methods has been assumed to be the same as that of the supplied [3H]thymi-dine. As the concentration of [3H] thymidine is increased from low values (i.e. less than 10-6M) the specific activity of the [3H]dTPP pool rises and so does the incorporation of radioactivity into DNA. This is at a time when the true rate of DNA synthesis remains unchanged. Thus the proporation of the dTTP pool which is radioactive, ([3H]dTTP(dTTP + [3H]dTTP)), is equal to the proportion of DNA thymine which is radioactive ([3H]thymine/total thymine). This equation may be rearranged to give  

This experiment can be done with coverslip cultures or directly in a 24-well TC tray. 

Wittes and Kidwell (1973) have described a kinetic approach to measuring the pool size of dTTP which involves growing cells continuously in 10/iM tritiated thymidine which they found to label about 20% of the DNA thymine residues. They calculated that at this concentration 106 L929 cells growing in suspension would convert 9.5 pmol extracellular thymidine into dTTP per min during S-phase. 

None of the methods is totally satisfactory. The first two involve addition of agents designed to alter the cell metabolism and the third 

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