I am trying to synchronize my cells at G1/S with thymidine double block, 2mM final conc., 18h each block, with 8h release in between. I collected cells at different time points (starting from T=0h), and analyzed their cell cycle using FACS (PI Staining). [attached data]
The cells proceed somewhat in sync but are not completely synced at release. Can anyone advise me on what parameters I should focus on optimizing? Thymidine conc. or blocking time or something else? Really appreciate the help!
Hi.!
What cells are you using? And did you mange to have a good S phase block with double thymidine block? I couldn't see T=O hr in the picture attached. Please take a look at the attached paper once.
Thanks
Satish
Dear Satish,
I am using mammalian cells, and the second picture in the left most column is T=0h
Thanks
Hi Ayon,
I am sorry but I don't think synchronisation at early S phase using double thymidine worked at all (looking at T=0 hour). Is it possible that you just need to replace your thymidine stocks?
Thanks
Satish
Hi Ondrej,
I am trying to optimize the sync as per your suggestion, and I collected cells every 2hrs after the first release. Based on the attached data, at what time point after the first release would you recommend to start the second block? Please note that due to uneven cell numbers the staining is not even, so the graphs are not well aligned with each other. However, in most of them the shape is clear.
Thanks a lot for your help.
Ayon
Hi Ondrej,
Thanks a lot for your suggestions. They’re very helpful, much appreciated.
I have one more question. My experimential design requires me to trypsinize and seed cells right after release. How well do you think the cells will remian in sync after trypsinizing?
Thanks again.
Ayon
Hi Ondrej,
Sorry to bother you again. I have tried doing the second block after 12h from release, Is it a bit odd that single block looks better than double block?
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