Double-Thymidine Block for Hela Cells - Growth rate (Oct/06/2005 )
I am trying to do a double-thymidine block of Hela cells using a concentration of 2mM thymidine.
I want to follow the expression of my protein every hour, for 12 hours, after release through the cell cycle.
Most protocols are pretty typical in that....
1. Plate cells anywhere between 20-40%
2. Add to 2mM thymidine for 19 hours
3. Release into fresh media for 9 hours
4. Add to 2mM thymidine for 16 hours
5. Release into fresh media and sample for 12 hours
My concern is that the cells seemed fairly close to confluency when I started my sampling. Hela cells are contact inhibited and I'm thinking that this will screw up my cell cycle analysis ie. the background from the already confluent areas of the plate. Between 8-10 hours, during mitosis, there were certainly a greater proportion of cells rounded (in mitosis) but I'm thinking that it might not be enough to overcome potential background. I'll be using cyclins as positive controls in the Western blotting but I'm wondering whether its worth doing all that work? Has anyone done this protocol with Hela cells? Perhaps my particular culture might growing a little too fast? The thymidine is made the day of and 2mM is widely used in the literature.
Any suggestions?