I'm maintaining mouse PBMCs in 10% FBS for a LPS stimulation experiment. I'm going to plate the cells in 96-well plate and treat them with LPS for ELISA. Just wonder if you guys starve the cells before LPS stimulation? More generally, can anyone give some comments about cell culture starvation? I've seen some people starve the cells while others not. So under what kind of circumstances should we starve the cells? Or for what kind of end-points should we do that? Many thanks!
I usually starve cells for 24h prior to experiment, if i am looking in signalling cascades that can be activated with proteins from FCS. Thus, the point is just to lower basal (for example ERK-p, p38-P, RhoA-translocation...). Depending of type of experiment, I sometimes starve cells for gene expression studies.
You have to be careful with primary cells, since some don't like to be completely deprived from FCS. so they might need to be in 2% FCS or charcoal striped FCS.
Hope this helps
Hi Danijela, I did know that when we need to look at phosphorylation, we have to starve the cells to lower the background. Anyway, your answer gave me a lot of confidence. Thank you very much.
I have never starved PBMCs for this assay but always use an FCS with ultra low endotoxin content. If you are using standard FCS you will get high background because it contains quite a lot of LPS already.
Why don't you do your LPS activation right away after sampling? Starvation is usuafly done for cell lines maintained in a medium that could interfer with the test. In culture, some PBMC subsets may die earlier than others and you may finaly work with a preparation that might not reflect the initial ex vivo sample. As said Danijela Markovic survival of some PBMC subsets in the absence of serum might be very low. As said Peter West, of course you have always to deal with FCS free of endotoxin contamination.
Thank you all for the suggestions and comments. Now I believe I have obtained a better understanding of why and when we should starve the cells.
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