I'm trying to synchronize MCF-10A cells with double thymidine block to enrich a centrosomal protein at G1/S transition following this protocol:
1) Add thymidine to final 2 mM for 12h (from a 100 mM stock solution made in PBS)
2) Wash cells 2x with complete media
3) Release cells in complete media for 8 h
4) Add 2nd round thymidine to final 2 mM for 12h
5) Wash cells 2x with complete media
6) Release cells in complete media and collect at 0h, 2h, 4h, 8h, 12h, 16h.
I did twice and got an enriched cell population with centrosomal staining only at 2h following release. That sounds a bit confusing to me, since I was expecting the same pattern at other time points, such as 0h and 4h.
Dear Luiz Silva,
For proper planning of cell synchronization, it is very important to know exactly the parameters of the cell cycle of the studied cell culture (duration of G1, S and G2 phases).
To do this, in parallel with the main experiment, it is necessary to examine cells using the inclusion of BrDU or more modern S phase labels.
You didn't specify which centrosome labeling you used? Which protein would you like to detect at the G1/S border or early S phase. You may find it helpful to read my review on cell synchronization to modify your synchronization protocol. For example, replace thymidine by hydroxyurea or afidicolin. The second of these reagents is more expensive, but more effective and has fewer unwanted side effects.
Best regards,
Rustem Uzbekov
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