I went through papers in which people had carried out thymidine block followed by nocodazole block for synchronizing cells in the G2-M phase. However, after the thymidine block for 24 hrs,a 3 hr gap is maintained before the nocodzole tretment.
What would be the consequence if this 3 hr duration is not followed and instead cells are treated with nocodazole immediately after the 24 thymidine treatment?
Thymidine blocks the cells in S phase, 3hr gap to enter the synchronized cells in M phase.
After the thymidine block, many of the cells will be synchronized in late G1/early S phase. The release for three hours allow the cells to proceed through S phase and fully duplicate and enter G2. The nocodazole treatment is supposed to block the progression from prometaphase (when the DNA is further condensing after NEBD and spindle attachments/alignment begin) to metaphase (the fully condensed chromosomes align along the metaphase plate). Nocodazole could be added without releasing the cells, but you will likely have increased cell death due to longer incubations in nocodazole (you will have to incubate for an extra three hours).
After the nocodazole treatment, most cells in my experiment became round and only loosly attached on the surface. Is it the same with yours or mine has gone wrong?
rounding of the cells and loss of attachment is normal cell behavior that precedes the cell division and since the cells are stuck in M phase they round and await the division.
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