Hi everyone,
I want to image mitotic phases cells, anyone can give me some suggestions? Do I need to synchronize cells to enrich more mitotic cells? I tried to synchronize cells to G2/M using RO-3306 and released 1 hours to fix cells for IF assay, but still not so many cells in Mitosis phase. Is there some side effects for RO-3306?
Thanks.
Hi, Weiyan
What I do is just to fix my cells and search for mitotic cells after immunostaining. I don't use chemical synchronisation to avoid any (possible) side effect on cell division progression. It takes longer to find dividing cells but in my opinion is better.
You can also try with mitotic shake-off. That way you get dividing cells without any treatment.
Regards
Hi Weiyan,
You can try to find unsynchronised mitotic cells but indeed will take a little while and lots of fields to be captured.
I already used nocodazole for G2/M arrest and worked very well! The cells were alive and were all synchronised, but I used for protein extraction for Western Blot, so I did not need for an specific phase. If you want to capture all the mitotic phases, you don´t need to synchronise, but I suggest that you plate the cells and wait for 24h or more dependent on the cell expansion rate so you can have better changes to find different phases of mitosis. If you synchronise and cells seem stressed you could try adding half of the used media (cells express a lot of growth factors in the media) and half of new media (so cells have new nutrients as well). This way is less stressful for the cell when you release from the treatment. You can also try thymidine block and release as it is less toxic than nocodazole, release, and fix the cells in different time points.
Good luck!!!
@ Raúl and Priscila. Thank your guys very much for your answer. I may try to use unsynchronized cells for safe.
Weiyan
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