After nocodazole treatment for cell cycle synchronization, most of my cells became round, but didn't float. What happened to them? Can I continue to lyse them for coIP and western blot?
What cells are these?
The cells appear to be dead.
Perhaps you need to titrate the nocodazole, and see what is the least effective concentration that doesn't induce cell death.
Thank you Sudeep Kumar , they are Hela cells. I agree that they might be dead, but strangely they didn't float. I'll try different concentrations!
THE CELLS APPEAR TO UNDERGO APOPTOSIS. THEY ARE SHRUNKN BUT STILL ATTACHED ..
You can stain them with simple stain like H&E and evaluate them for apoptosis.
This dose may be a new method for induction of apoptosis in cancer cells.
Mohamed Elkablawy That's interesting. Because most cells after this treatment looked just like that. I treated 13 plates and it happened to them all.
To my knowledge, nocodazole will sync the cells by doing prometaphase arrest and the mitotic cells appears round. The answers to your question on lysing them for coIP and western blot actually depend on your purpose of doing the cell sync. If you need the floating cells, you can do mitotic shake off to your plates and collect them for further processing. Hope this helps!
Ian Timothy Sembiring Meliala Thank you. But I'm not sure whether they are mitotic or apoptotic cells. Should mitotic Hela cells resemble this? Because to me, they look like cells after trypsinization; they are similar both in shape and size and are also weakly attached to the substrate.
Cells after nocodazole block appear like this. Even when the cells are asynchronous, you should be able to see ~5% of the cells rounded as they are undergoing mitosis.
If you want to look at mitotic cells only, you can collect the medium from the blocked cells, hit the culture vessel from the side vigorously to detach the blocked cells and collect them by gentle pipetting on the surface.
If you want to make sure the cells are still alive, you can check the cells under higher magnification to see if they are still spherical (rounded, dark center and bright smooth edge). If the cells are dying, most of the time they are not spherical but deformed.
Man Hei Cheung Thank you so much! This is very helpful! Now I know that those rounded cells are actually undergoing mitosis!
Stopping the cell cycle in mitosis usually triggers the process of apoptosis in the cells. Cells in the process of detaching from the surface of the culture vessel.
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