Nocodazole, cell cycle arrest in G2/M - (Aug/27/2008 )
Dear All,
I am going to arrests my cells which are African green monkey kidney cell line in G2/M using Nocodazole. I have to optimize the concentration and time of application of this chemical. I did start with three concentration of the chemical 40, 100, and 200 ng/ml. But it turned out that Nocodazole even in the lowest concentration is toxic to my cells and killed most of them. I was wondering if anyone has any ideas or references about using Nocodazole. I am so desperate and do appreciate your help.
THNX
Haki 
I am going to arrests my cells which are African green monkey kidney cell line in G2/M using Nocodazole. I have to optimize the concentration and time of application of this chemical. I did start with three concentration of the chemical 40, 100, and 200 ng/ml. But it turned out that Nocodazole even in the lowest concentration is toxic to my cells and killed most of them. I was wondering if anyone has any ideas or references about using Nocodazole. I am so desperate and do appreciate your help.
THNX
Haki
Haven't worked with the compound, but can't you first try lower concentrations? Do a serial dilution op to less than 1/100 of a nanogram and if you then stille experience toxicity I would try a different cell line? How long did you incubate your cells with the compound? (12-18 hours should be sufficient for synchonisation according to wikipedia, don't know how reliable that is).
I use nocodazole on my CHO cells at 50ng/mL. I only incubate for 10 hours in order to keep the cells more adherent. I'm wondering if your cells are really dying or simply becoming non-adherent. Cells in mitosis round-up, come off the coverglass/well and look very much like dead cells. Do a google search with the name of your cell line and nocodazole to find papers where people have used these cells with this drug. See what concentration and incubation times they used.
Thank you for the responses guys.
I have been trying to synchronize my cells in G2/M, have looked at literature but nobody has used the cell line that I am using for cell cycle. I cannot use any other cell line because this is the only permissive cells for my virus which I am going to infect them later on. I also did a pilot study with the chemical and after application monitored my cells hourly till 10 hours. Cells started to come up at 7-8 hours post treatment.
I just was wondering how confluent cells should be to apply Nocodazole on? Also, is it possible that I use Thymidine to synchronize my cells at G1/S, then treat cells with Nocodazole for 10 hours. I appreciate any comments and data you may want to share.
Thank you so much
Haki