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... 

Variations and other combinations of these methods are obviously possible, e.g. mitotic selection and thymidine block aminopterin block reversed with low thymidine followed by a high thymidine block reversed with deoxycytidine. 

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 selection of mitotic cells has been described in 11.2 and the proportion of cells in mitosis can be increased by use of the mitotic blocking agents colcemid or preferably nocodazole. The combination of a single thymidine block for 15-16 h followed by a 4-5 h in 0.04/ig/ml nocodazole gives a yield of 25-30% when mitotic cells are harvested (Zieve et al., 1980) and further mitotic cells can be obtained with subsequent shakings. 

Synchronizing Cell Cultures - Laborotory procedure of manipulating cells to bring all of the them to the same phase of the cell cycle. It is accomplished by a thymidine block. 

Thymidine Block - Mechanism for synchronizing cells in which thymidine is added to cells to inhibit DNA synthesis. This prevents passage of the cells from the G1 to S phase of the cell cycle. Subsequent transfer of cells to a medium lacking thymidine reverses the inhibition and allows them to initiate DNA synthesis synchronously. 

A confluent flask should be washed twice in sterile PBS and the cells trypsinized in 10 ml of trypsin/EDTA solution. Transfer the cells into the roller bottle at a density of 1.5 x lO /cm and in a final volume of 400 ml complete medium. Charge the bottle with sterile filtered 5% C02/95% air and place at 37°C on a roller apparatus at a rotation speed of 0.5 rpm. After 2 days the bottle should be confluent. At this point replace the medium with 200 ml complete medium containing 2 vciM thymidine and incubate for a further 11 hr in order to accumulate cells in S phase. This 11-hr thymidine block is most conveniently carried out overnight. 

On completion of the thymidine block wash the monolayer of cells once with 50 ml complete medium and then return the bottle to the roller apparatus with 150 ml fresh complete medium equilibrated with CO2. After 4 hr add nocodazole to a final concentration of 600 ng/ml. The nocodazole may be stored at -20°C... 

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...

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