Double thymidine block

The double thymidine block is the method most frequently referred to in the literature but it is tedious to apply and suffers from the disadvantage that the cells enter S-phase with a pool of dTTP which is decreasing over the first hour or so and this makes estimation of... 

The G2-phase of the cell cycle is perhaps the most difficult to study as it is the most difficult phase in which to obtain a synchronised cell population. This is because, if cells are synchronised by selection at mitosis or accumulation at the Gl/S boundary, by the time they reach G2 much of the synchrony has been lost. This is because of the dispersion forces arising from the different rates at which individual cells in a population traverse the cycle. G2 populations are always contaminated with cells in other phases of the cycle and the maximum fractions of Chinese hamster (CHO) cells obtainable in G2 are 0.7 by double thymidine block and 0.4 by mitotic selection (Enger et al., 1968). 

The relative transcript levels and transcription rates of the polymerase gene during the cell cycle in HeLa cells were determined using cells synchronized by a double thymidine block. Total RNA as well as nuclei from cells were isolated at hourly intervals during the progression of S phase and examined either by nuclear run-off transcription or Northern analysis (10). 

Fig. I3a-c. Infection by viral RNA of untreated and polycation-exposed HeLa cells during different stages of the growth cycle. Suspended HeLa cells were synchronized by a double thymidine block (Tobia et al.,1970). Synchrony was measured by following...

All these methods succeed in accumulating 80-90% of the cells at the Gl/S-interphase and are very suitable for obtaining populations of S-phase cells for study. They are readily scaled up to the level of roller bottles though at this scale the amount of thymidine required for a double block is considerable (0.5 g/roller bottle). Concentrated solutions of thymidine can be made up and sterilised by autoclaving but most of the other solutions should be filter sterilised. 

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