Double-Thymidine Block protocol

Dan Levy's Lab 2018

Michal Feldman

Double-Thymidine Block protocol

This protocol is used for synchronizing cells to G1/S phase. Using two steps of thymidine blockage is useful for maintaining cells synchronized for 3 following cycles.

Reagents:

Standard Cell Culture Media (example: DMEM + 10% FBS)

50 mM Thymidine blocking solution (in PBSx1)

Sterile PBS or serum free media

Procedure

1. Grow cells in culture in standard media to approximately 40% confluency.

2. After the cell adhere, add 40ul of Thymidine blocking solution (final concencetration 2mM) for each 1ml of culture media.

3. Incubate culture for 16 hours.

4. Remove the media and wash culture 2x with sterile PBS.

5. Add fresh cell culture media (containing serum) and incubate for 9 hours.

6. Again add 40ul of Thymidine blocking solution for each 1ml of culture media.

7. Incubate for an additional 14 hours.

8. Remove the media and wash culture 2x with sterile PBS.

9. Add fresh culture media.

10. At this point, cells will be at G1/S phase and are ready to be released into cycle over the next 15-20 hours.

11. The cell cycle position of the cells can be validated by propidium iodide staining and flow cytometry, or by Western Blot, using appropriate markers.