Endotoxins-free FBS - (Jul/12/2012 )

Hi,
I am experiencing problems when culturing primary monocytes. After I use LPS to stimulate them and asses for differences in gene expression between stimulated cells and mock, changes are either similar or in mock even bigger. I decided to rest the cells for 24 hours first and do stimulation after this time, but no change. So, I think there may be endotoxins in serum I use (RPMI 1640 + 10% FBS - Gibco). However, I am not able to find any producer who offers endotoxin-free or at least endotoxin-low serum. Does anyone of you have any suggestion?
Thanks a lot!
Paja

Paja on Thu Jul 12 15:12:28 2012 said:

Dear Paja,

All serum comes with a certicate of analysis. This is a document with many test results i.e. Haemoglobin, endotoxin, mycoplasma etc.

We buy serum with low endotoxin levels which we aslo batch test. NO SERUM IS ENDOTOXIN FREE!!!!!!!!

Also the country of origin is very important because the quality of serum is different from each world region i.e.

New Zealand>(better than) Australian>North American>South American>European>British

The price of the serum reflects this i.e.

£230.....£180......£150........£100.............£80........£50

I hope this is useful

Kindest regards

Uncle Rhombus

As usual, Uncle Rhombus to the rescue with the gold standard for advice

So I'm just going to add a "what he said" and then a question....

Why have you decided that the FBS is the cause of the problem?

Dear Uncle Rhombus and leelee,
Thank you both for your replies. First, I do not know if the FBS is the reason of my problems, but by a process of elimination I came to serum now. First, changed culture plastics, used brand-new media and FBS and other chemicals for cultures, new aliquots of LPS. I donated my own blood draft into vacutainers (EDTA) to eliminate potential effects of plastic bags used for buffy coat preparation at the Transfusion Unit (T.U.) at out hospital (all optimizations we are doing on material from these T.U. and we do not know what chemicals may be inside the bag). I performed 24 hour resting but no change, still the mock levels of inflammatory cytokines differ from time 0 over 2, 4, 8, and 24 hours of cells cultuting (I expected them to be even as no process of stimulation occures here) a frequently they reach or even overcome the levels detected in LPS-stimulated cells. So, something is going on in these in vitro cultures when mock is stimulated evenly as positive cells. :-( I do not want to think about what alese I have to test/change in my experiments to figure the problem out. It is like a nightmare - so many people publish data from monocyte-based experiments and I can not move on! :'-( So I look for serum with the possible lowest endotoxins I can find...

I had no idea there is a difference in quality across the world, thank you for this very useful info.

Paja