get metaphase cell by nocodazole? - (Apr/01/2005 )
hellow everyone,
I want to get metaphase cell.
Protocol i used: Nocodazole (final concentration 50ng/ml) was add in the culture medium for 18-20h. But no more(just as not treated ) metaphase cell could be found, and many cell become round and their nucleus contracted -indicated they are dead or almost dead.
What is the problems?
Any suggestions are welcome!
thanks in advanced!
-bluemoon819-
QUOTE (bluemoon819 @ Apr 1 2005, 08:02 AM)
hellow everyone,
I want to get metaphase cell.
Protocol i used: Nocodazole (final concentration 50ng/ml) was add in the culture medium for 18-20h. But no more(just as not treated ) metaphase cell could be found, and many cell become round and their nucleus contracted -indicated they are dead or almost dead.
What is the problems?
Any suggestions are welcome!
thanks in advanced!
I want to get metaphase cell.
Protocol i used: Nocodazole (final concentration 50ng/ml) was add in the culture medium for 18-20h. But no more(just as not treated ) metaphase cell could be found, and many cell become round and their nucleus contracted -indicated they are dead or almost dead.
What is the problems?
Any suggestions are welcome!
thanks in advanced!
what's the doubling time of your cells, what are your cells?
you may need to perform a titration of nocodazole. I am not too sure what 50ng/mL is with respect to what we use which is 100nM concentration for 12 hours.
When you enhance with nocodazole and if you are working with fibroblasts, because they are halted in metaphase they can be easily dislodged, so save the media when trysinising as that is where most of the cells may be!
Good luck
Nick
-methylnick-