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cell culture.....help
Posted by: keysia (IP Hidden, Unregistered user, )
Date: April 29, 2005 08:31PM
hai my work involves treating cells (cell line) with cytokines and checking the upregulation of certain genes by real time pcr.
the problem is that.....when i try to replicate my experimet , the fold increase over my control (untreated cells) seems to vary from 5 - 25 each time. Though there is always an upregulation the fold increase seems to vary...is this normal. I have used the same media for all the experiments . please help.!!!!!!
Re: cell culture.....help
Posted by: 5'-ATCG (IP Hidden, Unregistered user, )
Date: May 2, 2005 05:24PM
It's a pretty common phenomenon related to the unstable and inconsistent activity of growth factors and/or cytokines. Remember to use fresh cytokine from your freezer (aliquotes) each time and avoid any freeze-and-thaw cycle. Having say that, some cytokines from certain vendors are very sensitive and easy to degrade......so make sure to purchase these items from a relaible and qualified source, my lab has been using growth fcators and cytokines from Epoch Biolabs (for 3 years now) with high satisfaction.
Re: cell culture.....help
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 12, 2005 12:10PM
if possible you may wish to check the stability and degradation of cytokines from multiple suppliers to see which might yield the best results.
Re: cell culture.....help
Posted by: U. Penn (IP Hidden, Unregistered user, )
Date: May 21, 2005 02:51PM
I have excellent experience with IL-3 and IL-6 from epoch bio. In my hands, they induced STAT-1 and STAT-5 nuclear translocation at 1 ng/mL (20 min treatment) everytime.
I think it all depends on the stability of your cytokines in which medium at what temperature.
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