Double-Thymidine block of Hela cells - (Mar/02/2007 )

Hi everyone,

I have been doing Hela cell arrest in G1/S phase, followed by release to profile the expression of my protein of interest during cell cycle. I referred the paper which shows the same experiment. I used the same condition as they used. Grow Hela cells to about 60-70% conflucence, then 18h Thymidine block 2mM, then release into Dextran coated Chacoal (DCC) treated media which removes the small molecules in the media for 9h, then secondary thymidine block for 17h, finally release cell into fresh media 0-12h. I analyzed cell by FACS-PI staning and found that most of the cells moved fowards the cell cycle after G1 release: from G1-S-G2/M within the 12h process, but there is a population which seems to be sustained or still arrested in G1 and before release, cells are not completely sychronized (still a population in S/G2/M, but not as many as the normal control). The only difference is that after the first 18h blockm, they released cells into thymidine-free media, but I just used DCC media. I am not sure DCC helps to remove the thymidine in the media, but would that makes that much difference. Which media do I need to use potentially. will is really help to sychronize all cells in G1. Dialyzed serum might be the media to be used, as somebody told me. All experts here, have some recommendations?

Thanks a lot in advance!!


Nan

[quote name='nan820913' date='Mar 3 2007, 01:37 AM' post='89847']
Hi everyone,

I have been doing Hela cell arrest in G1/S phase, followed by release to profile the expression of my protein of interest during cell cycle. I referred the paper which shows the same experiment. I used the same condition as they used. Grow Hela cells to about 60-70% conflucence, then 18h Thymidine block 2mM, then release into Dextran coated Chacoal (DCC) treated media which removes the small molecules in the media for 9h, then secondary thymidine block for 17h, finally release cell into fresh media 0-12h. I analyzed cell by FACS-PI staning and found that most of the cells moved fowards the cell cycle after G1 release: from G1-S-G2/M within the 12h process, but there is a population which seems to be sustained or still arrested in G1 and before release, cells are not completely sychronized (still a population in S/G2/M, but not as many as the normal control). The only difference is that after the first 18h blockm, they released cells into thymidine-free media, but I just used DCC media. I am not sure DCC helps to remove the thymidine in the media, but would that makes that much difference. Which media do I need to use potentially. will is really help to sychronize all cells in G1. Dialyzed serum might be the media to be used, as somebody told me. All experts here, have some recommendations?

Thanks a lot in advance!!


Nan
[/quot]
accord to ATCC guidence, hela cell should be cultured in DMEM media,and the first block should be done at 25-30% confluency of HeLa cell culture

MCB Vol. 13, Issue 6, 1977-2000, June 2002, Michael L. Whitfield et al.
this paper would be helpful

Thanks for the paper and the concentration of the cell you suggestted is helpful. By the way, I did culture my cells in DMEM, it is just mixed with DCC treated serum to insure that the thymidine dose not come in from the serum. I just wonder is DCC serum really helpful to strip the thymidine or do I need to buy some turely thymidine-free media.