Does anyone know where to find the list of average concentrations of hormones in FBS? I know that insulin is about 40 pM in 5% FBS, but what about the other hormones?
And does anyone have a protocol how to remove insulin from FBS? (affinity purification?)
Charcoal-stripping is a not option for me since it removes also other hormones and compounds.
We actually had a batch that of FCS, which we had charcoal stripped, sent for analysis. Here is a table of the hormone levels of FCS stripped and unstripped:-
Table1.
Effects of charcoal stripping on hormone levels of calf fetal serum
Hormone Progesterone/Testosterone/Estradiol/Estrone
Level ng/ml (nM)/pg/ml (nM)/pg/ml (pM)/pg/ml (nM)
FCS ust < 0.20 472 (1.6) 0.4 (1.5) 14,247 (53)
Stripped < 0.20 < 200 ND 13
Levels of hormones in unstripped/stripped fetal calf serum as determined by an independent laboratory, Texas Veterinary Medical Diagnostic Laboratory, College Station, TX.
Hormone levels are different in every lot. And the mean concentrations would tell you very little, since your experiments will likely employ one specific lot! So if serum insulin level is a major concern, I would encourage you to perform an ELISA-based measurement to assay for its concentration.
In terms, of insulin-depleted sera, there are some commercial suppliers, simply Google for "insulin-depleted serum." If you work with animals, there are protocols to suppress insulin production in vivo (e.g. treatment with somatostatin or streptozotocin), but I haven't seen ones to remove insulin in vitro. Perhaps you could deplete with anti-insulin antibodies, but I imagine this would be cost-prohibitive.
I'm not sure if primary cells (mature adipocytes) will be happy without serum.
There are SF media for a number of primary cell types. Here are some examples: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/serum-free-media/primary-cell-medium.html. I would try medium for fibroblasts or epithelial cells. If that does not work, you may consider using a defined FBS (HyClone has several types of it). Accompanying certificate of analysis will specify amounts of insulin and some other hormones. If you have some particular hormones in mind you should be able to measure their concentration by using ELISA kits or you can account for their interference by setting up appropriate controls in your experiments.
P.S. Amsbio claims it has serum-free media for adipocytes (http://www.amsbio.com/protocols/AdipocyteCareRV08.08.pdf). I do not have personal experience using their products yet.
We actually had a batch that of FCS, which we had charcoal stripped, sent for analysis. Here is a table of the hormone levels of FCS stripped and unstripped:-
Table1.
Effects of charcoal stripping on hormone levels of calf fetal serum
Hormone Progesterone/Testosterone/Estradiol/Estrone
Level ng/ml (nM)/pg/ml (nM)/pg/ml (pM)/pg/ml (nM)
FCS ust < 0.20 472 (1.6) 0.4 (1.5) 14,247 (53)
Stripped < 0.20 < 200 ND 13
Levels of hormones in unstripped/stripped fetal calf serum as determined by an independent laboratory, Texas Veterinary Medical Diagnostic Laboratory, College Station, TX.
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