Hi, I am now studying a cell-cycle-dependent protein with HeLa and Hek293. I used double thymidine method for cell cycle arresting. However, I have found that cells (both HeLa and Hek293) seemed to become irregular after first round of thymidine treatment. (As the figure shows, suspected to be apoptosis?). I wonder whether it is a frequently observed phenomenon. Is there any way to prevent it? Thank you!

Protocol details in my case:

1. 2mM thymidine in ddH2O

2. Confluency is around 30% to 40% before first round of thymidine treatment

3. Cells are grown on glasses

4. The photos are taken 22h later after first round of thymidine treatment