I'm planning to synchronize Hela cells at early S phase using double thymidine block. I've read different protocols, suggesting different time periods for the incubation of cells after the thymidine treatment, i.e., for 12, 16 and18 hours, and afterward the release for 9 hours, then repeat. Is a 18-hour blockage time stricter and better for the arrest? Will a longer blockage bring more unwanted side effects on the cells? What do you recommend?
Also, I read that I should conduct the experiment when cells are at about 40% confluency since cells will keep dividing during the double treatments, however, a senior student adviced me to begin with cells at 70% confluency. I thought the overcrowdedness of the cells might reduce the block efficiency. Could you please give me some suggestions?
A third question is regarding serum deprivation. I once read that it can't be applied to Hela cells. Then from different sources, people say that it can arrest Hela cells at G0/G1. Can someone help enlighten me on this?
A point to add is that in my lab, students don't release cells with deoxycytidine, just DMEM with 10% FBS. Is deoxycytidine neglectable there?
Many thanks to your experts' kind help!
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