endotoxins-free FBS

[Paja]

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 improvement. 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

[CrowSan]

Hmm. have you considered serum starving the cells 24 hrs prior to stimulation? This may help. I think there are also serum substitutes on the market that replace the FBS with a non-animal derived source (a reconstituted FBS). I'd try serum starvation first though.

[mchlbrmn]

The suppliers in my area sell different grades of serum, with endotoxin levels as one of the things they test, and give written certifications of the maximum levels. They also will send samples from specific lots that can be tested in your assay before you commit to a large purchase.

[Paja]

Hmm. have you considered serum starving the cells 24 hrs prior to stimulation? This may help. I think there are also serum substitutes on the market that replace the FBS with a non-animal derived source (a reconstituted FBS). I'd try serum starvation first though.

Hi CrowSan, thank you for your suggestion. I haven't considered FBS starvation because I thought monocytes die without complete serum withdraval. Maybe I shall try.
Paja

[Paja]

The suppliers in my area sell different grades of serum, with endotoxin levels as one of the things they test, and give written certifications of the maximum levels. They also will send samples from specific lots that can be tested in your assay before you commit to a large purchase.

Hi mchlbrmn,
could you provide me names of some of suppliers from your area please? Thank you!
Paja

[CrowSan]

A quick "peek" at the literature indicates a lot of people serum starve overnight prior to LPS activation. Try "googling"
"monocyte LPS stimulation serum starvation" to see if you can get a protocol for this.