In your opinion, which E. coli LPS strain should be selected in order to analyze a good IL-10 in vitro production by BM-DCs?
Sigma-Aldrich commercializes at least 6 different LPS strains: O111:B4, O127:B8, O128:B12, O026:B6, O055:B5, and K-235. What about the amount of DCs (24 wells plate), concentration of LPS, and read-out time point?
Is your choice also suitable for stimulating human DCs (generated in vitro)?
Hi,
Are you sure IL-10 would be induced?
LPS (any of the above for my knowledge) is known to induce high inflammatory response, via TLR4, which IL-10 is one of the factors secreted (in Treg for example) to suppress.
Dear Ehud:
Thank you very much for your commentary.
About your question, it is known that, as a homeostatic effect, DCs stimulated with inflammatory stimuli can produce IL-10; on the other hand, "tolerogenic DCs" (tDCs) produce higher amounts of this cytokine.
I have already performed several experiments on DC stimulation (in vitro), obtaining IL-10 from LPS-stimulated BM-DCs; nevertheless, the amount of this cytokine was usually low. In contrast, DCs stimulated with a heat-killed Enterococcus faecalis strain (another "inflammatory" stimulus), generated higher amounts of IL-10. I still have to stimulate DCs with a tumor cell line, in order to generate tDCs...
I employed O111:B4 LPS in my experiments, but since usually there are strain-specific effects, I was trying to get some information about this phenomenon.
Thanks again for your contribution.
Best regards!
Hi Matias, this is our experience with BM-DC from C3H /HeN mice. We use 10 exp 6 DC per ml in 24-well plates (Nunc), and culture them with 10 μg/ml of LPS (Escherichia coli O26:B6; Sigma). In 24 hs supernatants we detect aprox 275 pg/ml of IL-10 (Sandwich elisa R&D). (Ref, Poncini et al. Infect. Immun. June 2008 vol. 76 no. 6. 2633-2641).
We have not tested the other LPS strains from your list. We tested BM-DC from Balb/c mice with similar results.
Hi Catalina:
Thank you very much for your answer.
In our case, we stimulate 4x10(5) BALB/c BM-DC/ml in 24-well plates (GBO) with 1 ug/ml of E. coli O111:B4 LPS (Sigma-Aldrich). After 24 h, we observe similar amounts of IL-10 (approx. 200 pg/ml, measured by BD OptEIA ELISA Set). Nevertheless, DCs produce much higher amounts of IL-10 (2.000 pg/ml) when stimulated with a heat-killed E. faecalis probiotic strain in the same conditions (unpublished data).
Apparently, these two LPS strains seem to have similar effects... Does anyone have a different experience?
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