I am doing thymidine double block in 293T cells to arrest cell cycle in G1/S phase. But the results in FACS were not expected and only around 20% of cells stay in G1 phase. The protocol I followed is: (1) seed 4X105 cells per well in 6-well plate and incubate overnight, (2) add thymidine for 2mM final concentration and incubate for 18 hours, (3) remove medium, wash with PBS, and add fresh completed medium releasing 9 hours, (4) add thymidine again for second block and incubate for 18 hours, and (5) replace with fresh medium and collect cell in different time point (0, 6, 8, 10, 24 h).
Since the cell didn't blocked by thymidine even in time 0, I think probably I did something wrong in this process or even the thymidine solution. I prepared 400mM thymidine in PBS. Since it is difficult to dissolve even after incubating in 37 degree, so I heat it in 50 degree. Does anyone has experience with preparing this solution? Did I make any mistakes in making this solution or double block procedure?
Hi Yuhang,
your treatment protocol is fine. I would only recommend to wash cells directly with medium instead of PBS, in my case they then recovered better from first block or alternatively, you can extend release for up 10-11 hours to give all cells time to go through the S-phase.
Regarding preparation of thymidine stock solution, if I remember correctly, the maximum concentration I could achieve was 200 mM and it can indeed take some time at 37°C to be fully dissolved. When such stocks were stored at 4°C, sometimes crystals of thymidine precipitated, and those were even more difficult to re-dissolve. I solved this problem by preparing relatively diluted stocks of only 100 mM, dissolved directly in medium (without FCS). Medium instead of PBS was used to avoid too strong changes in culture medium after addition to the cells.
By the way, excess of thymidine inhibits the elongation of replication, so mechanistically, the cell cycle is blocked not at G1/S, but in very early S phase. Depending on the purpose of your experiment, you may also need 2h or 12h time points, 24h might already be too late because often the cells don't remain "synchronized" for so long time.
Good luck!
Eugen Werwein Thank you so much. I made new stock solution with 100 mM and I am starting new experiment according to your suggestion.
You should dissolve it in water to final conc. Of 50mg/ml at room temp.
- Badges
- Science method