In a culture of Bone Marrow Derived Macrophage (BMDM) the supplementation of the medium with 10% of Bovine Fetal Serum (BFS) inhibits the detection of IL-1β by ELISA, but not of other cytokines.
This problem can be solved by supplementing the medium with just 1% of BFS. Evidence suggests that the IL1β production is not inhibited, but rather the release.
Does anyone have an explanation for why this occurs? What is the mechanism of inhibition of IL-1β by BFS?
I hope you don't mind if I get the most important thing straight at the beginning:
FBS does not inhibit IL-1ß cleavage or release.
There are several possible explanations for your results:
a) your ELISA antibodies are inhibited by FBS - this is the case for some antibodies - solution: buy IL-1ß ELISA Sets from BD, they definitely work
b) Your incubation might be too long: the cells in 1% FBS starve and die, releasing pro-IL-1ß into the supernatant. your 10% FBS cells survive and nothing ends up in the media. the only reliable readout for real (cleaved) IL-1ß release is western blotting
c) your FBS is contaminated with weird stuff. get some from another batch or a different company and see whether it helps
Good luck and best regards,
Thomas
I hope you don't mind if I get the most important thing straight at the beginning:
FBS does not inhibit IL-1ß cleavage or release.
There are several possible explanations for your results:
a) your ELISA antibodies are inhibited by FBS - this is the case for some antibodies - solution: buy IL-1ß ELISA Sets from BD, they definitely work
b) Your incubation might be too long: the cells in 1% FBS starve and die, releasing pro-IL-1ß into the supernatant. your 10% FBS cells survive and nothing ends up in the media. the only reliable readout for real (cleaved) IL-1ß release is western blotting
c) your FBS is contaminated with weird stuff. get some from another batch or a different company and see whether it helps
Good luck and best regards,
Thomas
I have to agree with Thomas. I have never ever seen or heard of 10% FBS inhibiting IL-1B (or caspase-1) release.
However, one other possibility comes to mind: what stimuli are you using? ATP, Nigericin and the various crystalline stimuli are no problem at all in 10% FBS. We use BD Elisas, and BMDMs often produce more than 10ng(!) cleaved IL-1B with those stimuli. However, if you're transfecting, for example with dsDNA as an AIM2 stimulus, then FBS can definitely disturb liposome formation and lipofection. In that case, I would use optimem.
It may also be, as Thomas said, that the problem is with your antibodies. I can only think of two possibilities:
One is one of your ABs has a preferential affinity for pro IL-1B rather than the cleaved form. Then you may have to replace the AB and/or the kit.
The other is, of course, compatibility with FBS. If that's the problem, then you can simply do the experiment in serum-free medium and drop the 1% FBS completely. However, that also means that you will have to keep your experiment under 12 hours, so that your cells don't starve.
Good luck!
Eva
Concerning this experiment, the BMDM were infected with Brucella abortus.
Just like myself, other colleagues that did BMDM infection with B. abortus had the same problem with the detection of IL-1β when the medium was supplemented with 10% BFS, even using ELISA sets from BD.
B. abortus can penetrate cells in areas of lipid-rafts.
If it is true what Eva Bartok said above, that "FBS can definitely disturb liposome formation and lipofection". Would it be possible that the FBS is inhibiting the infection of B. abortus by this route?
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