G1/S cell synchnronization using double thymidine block

Thursday, January 29, 2004

Description
This protocol is designed to synchronize HeLa cells at the G1/S border (actually all G1). It is highly effect and gives almost complete synchronization in the culture. Note that this protocol will not work on cell lines with intact p53 apoptotic responses.

Procedure
1. Grow HeLa cells in standard DMEM +10%FBS to about 40% confluencey.
2. Simply add thymidine that has been resuspended in PBS to a final concentration of 2mM in the media.
3. Incubate culture at 37C for 19hrs (strict time keeping here).
4. Remove media and wash cells with PBS 3X.
5. Add fresh media without thymidine and let incubate for 9 hrs at 37C.
6. Add thymidine again to a final of 2mM and allow to incubate for another 16hrs.
7. Wash cells with PBS and add fresh media. The cells are now in G1 and will be "released" to progress through the cell cycle over the next 15hrs. The cells should be uniform for about 1-2 cell divisions and then regain their asynchronous state.

Recipes
Standard Gibco or Invitrogen DMEM is sufficient.
Any company FBS is fine.
Sigma thymidine powder is fine.

Supplies
Standard tissue culture supplies will be sufficient.

Tips
No need for filter tips here.

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G1/S cell synchnronization using double thymidine block