Hi
I am working on a panel of pancreatic cancer cells studying the effects of cytokines on cell behaviour. BXPC3 especially grow well in RPMI media with 10% serum, however when I grow them on serum free, they die within 24h and during this time they form tubes. Because I am comparing the effect of the cytokine between different cell lines, I have to use consistent culture conditions. I found proliferation assay is done in serum-free in many publications.
So my question is it necessary to starve the cells? or as long as the cells are happy and I have control it would not be an issue?
Thanks
Its better to starve the cells for at least a short period of time (4-6 hours). Otherwise if the cells don't even tolerate this short starvation, try lowering the concentration of FBS to 0.5%-1% before stimulating with your cytokines
Serum starvation is recommended to :
1. Shut down any signaling from receptors that may have been activated by cytokines and growth factors in serum (FBS) .
2. To allow recycling of down-regulated receptors to re-express at cell surface.
3. And allow signaling molecules that have been activated to come to a basal state - especially if these are the ones you are going to follow after cytokine stimulation. Otherwise your "baseline" will be high and you MAY not see your cytokine signal.
As Richard says, some cells -especially cancer cells -may not like serum starvation for long periods, and then lowering FBS to 0.5% may work. Test this before losing your cells!
Good luck !
It is important to add always a minimum of serum in medium for the cells. I would suggest 1-3% serum in medium as serum starved medium.
Regards
There is no simple answer to your question. Many people assume that all cells respond to serum starvation the same way, which is unlikely. Some considerations: Adding additional glutamine may help with some cells when working in serum-free conditions. Some people use BSA in serum-free culture. However, If you are testing the effect of lipid compounds on your cells, the delivery of the lipid compound in the presence or absence of serum would be different, thus the cellular response. If you do use serum or BSA, you may want to use charcoal-extracted material. Frankly, lot of tissue culture methods are based on faith-based research, like choosing the culture medium for the cells. You have to characterize the conditions specific for your cells.
In my opinion it is better to starve cells for at least a short period of time (10-12 hours), or if it impossible lowering serum concentration to .5-1.0%.
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