Hi Rafiou, actually is not weird what you're observing, it's common when you stimulate either PBMCs or BMDMs with LPS in vitro. There are several evidences in literature showing that CD86 surface expression is suppressed during LPS tolerance, and is not further inducible following recovery from LPS tolerance. I'm pretty sure that with 12h you're inducing tolerance on BMDMs. It's a matter of time (and even concentration), let's say a combination of them! Attached you can find just a recent experimental paper on that. What about your LPS? Is it from E.coli or other strains? Moreover, a recent paper have shown that CD86 expression decreases in an IL-10-concentration-dependent manner in B-cells (see attached paper and corresponding references). The effect of exogenous IL-10 on the expression of CD86 was previously demonstrated in monocytes and DCs. Furthermore, the autocrine effect of IL-10 on CD86 down-regulation was reported in human DCs following LPS stimulation. I hope this will help you!    

http://www.jimmunol.org/content/192/1/427.full.pdf

http://www.nature.com/articles/srep20044

Hi, you must check the differentiatios conditions... You will probably activated the cells before the LPS stimulation, and you could have "the final" effect of activation. Are all the reangent LPS-free? how is CD80 or CD83 expression?

Hi Juan,

Thanks for you answer. Yeah all my reagent are LPS-free. I doin't think that I have a pre-activation condition.

Hi Damiano!

Thanks for your clearly response!

All the best for all.

after 12 hr of stimulation you probably overwhelmed their ability to respond and they are on their way to apoptosis. Best results are achieved with BMNC or PBMC derived cells cultured for 4-5 days with GM-CSF and then stimulated with LPS for no longer than 6 hours.