Dear Cheng,

I usually perform this kind of experiments in HeLa cells to study the variation of several proteins during the cell cycle and especially mitosis. For me, the problem is due to your transfer step. Because mitotic cells are pretty sensitive, I imagine your cells don't appreciate a re-seeding step during mitosis and die. I don't know why you need to transfer your cells but I think it would be better to avoid it. Is it an important step for you ? What do you need to do with your mitotic cells after their collection ?

Best regards,

Guillaume Andrieu

Dear Guillaume,

Many thanks for your answer. Basically, the problem you mentioned is exactly the problem I have now----most of those cells just won't attach to the dish.

I transfer cells from one dish to another in order to harvest cells for IF and WB at specific timing after releasing from nocodazole. Do you have alternative means to achieve that? If there is any, would you please give me some advises?

Best regards,

Cheng

Dear Cheng,

When I realized this kind of experiment for IF, I put a coverslip directly in my petri dish so I can use it for IF without reseed just after nocodazole release. Just pick out the coverslip and fixate the cells or whatever you want. And I harvest the remaining cells for WB analysis.

Best regards,

Guillaume