thymidine block - (Dec/08/2011 )

Hi All,

Using Thymidine @ 2.5mM to block Hela cells at G1/S boundary prior to lysis. Its my first time using the chemical and I didn't notice any morphological changes to the cells after 20 hrs. I've used HU before for this purpose and the cells definitely looked different after incubation. Am I doing something wrong or is this normal?

Thanks!

Have you put them through a FACS or used some other method to determine the G1/S arrest? If not - do that first, I would suspect that the HU is much more toxic than thymidine, so the morphological changes you saw were related to that rather than the arrest.

I haven't done flo-cyt or FACs on my cells yet because I just started using the thymidine-- I am doing a western later this week though, so if it worked, I should see a difference in a few markers vs ascyronized control plate. And as for the second part, you might be absolutely correct -- I stopped using HU because it does nasty things to the cells (other than resulting in G1/S arrest). It makes sense the odd appearance of the cells could be related to that.

If anyone has experience with thymidine please let me know.