Hi Yuhang,

Double-thymidine synchronizations can be tricky. Could you answer a few technical questions below?

Do you know what is the confluency of your cells before you start the thymidine treatment?

Did you double-check that the thymidine was completely dissolved?

Did you analyze by flow cytometry after the first thymidine block? The %G2 peak should decrease after the first block (and indeed the %S peak should actually increase a little), while the %S peak should decrease after the second block as you mentioned previously.

Also, I assume you fix your cells prior to 7-AAD staining? What is your fixative? Are you gating away doublets before you gate using the 7-AAD channel?

You may have to optimize the timing of thymidine block/release for your exact clone; have you tried different incubation times such as 16 hours versus 20 hours? You could start with testing the first initial block at various durations just to confirm you're properly removing the G2/M peak (see above).

Another control you can consider doing is a thymidine-nocodazole block which should give you a G2/M arrest and thus should look very different than your double thymidine experiment.

Let me know if these questions do not make sense.

Thanks,

Barry

Barry Zee Thanks you so much for your help. Here are my answers for your questions.

(1) Confluence is around 50%. I seeded 300,000 cells per well in 6-well plate, and added thymidine 24 h after seeding.

(2) I think I dissolved the thymidine in a proper way, which was dissolved in PBS and it is a clear and transparent solution.

(3) Yes, I did. After 1st block, the %G2 decreased just little bit, from 14% to 12%, and %S increased from 55% to 71%. But after 2nd block, the decrease of %S and increase of %G1 were not striking.

(4) I fixed cells by using 70% ethanol on ice for 1 hour. The staining buffer I used was PBS+1%FBS+0.25%TritionX-100. I gated single cells and also 7-AAD positive cells (unstained control).

I didn't optimize my incubation time since I didn't observe any increase of %G1 after double block. I did nocodazole control, which worked perfectly in my other cell lines like MCF-7, a breast cancer cell lines, causing cell arrest in G2 phase. But it caused G1 arrest in my 293T cells. I attached my flowjo file here just in case if I analyze my data in a wrong way.

Hi Yuhang,

OK, so the majority of 293T cells accumulated at S-phase after the first block (18 hour incubation). You then release your cells for 9 hours. Did you look at the cells after the 9 hour release and before the second block? If the cell distribution looks the same after the release, namely most of the cells are still in S-phase, it could be that there is residual thymidine in your media (which may mean you need to do more washes or allow a longer release).

Also, have you tried seeding at a lower density such as 1.5E5 cells/ml, allowing attachment for 24 hours, before starting first thymidine incubation? Relatedly, have you determined the doubling time of your 293T cells as an unsynchronized culture from different starting densities? Often changing the starting density can dramatically influence the growth rates, and thus responsiveness to metabolites such as thymidine.

Thanks,

Barry

Barry Zee Thanks for your comments. It's really helpful. I didn't look at those experiments as you mentioned above. But I would like to try. And is there anything wrong with my data analysis? Since it is the first time I performed this experiment, I am not sure if I am doing everything properly.