Bone marrow-derived macrophages were stimulated with recombinant IL-6 (R&D), IL-4, IL-10 (both from PeproTech) or E. coli-derived lipopolysaccharides (LPS O55:B5; Sigma). For pre-incubation experiments, cells were incubated with 50 ng/ml IL-6 for 12 or 24 hours and subsequently exposed to either IL-4 or LPS alone or in combination with IL-6 for the indicated time points. For antibody-mediated neutralization of IL-10, cells were preincubated for 1 hour with either isotype control (10 μg/ml Purified NA/LE Rat IgG1, #554682 BD Pharmingen) or anti-IL10 antibody (10 μg/ml Purified NA/LE Rat Anti-Mouse IL-10, #554421) and subsequently exposed to IL-6 (50ng/ml) or IL-10 (10ng/ml) in the presence of either isotype control or anti-IL10 antibody for 12 hours. BMDM were transfected with 600 pmol of siRNA oligos against Stat3, Il6ra or a nontargeting control (all LifeTechnologies) as previously described40 (link).
Protocol detail
Macrophage Polarization Signaling Pathways
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Corresponding Organization :
Other organizations : University of Cologne, University of California, San Francisco, Max Planck Institute for Metabolism Research, Baker Heart and Diabetes Institute, Max Planck Institute for Biology of Ageing, Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases
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Variable analysis
independent variables
- Recombinant IL-6 (R&D)
- IL-4 (PeproTech)
- IL-10 (PeproTech)
- E. coli-derived lipopolysaccharides (LPS O55:B5; Sigma)
- Incubation time (12 or 24 hours) with IL-6
- Isotype control antibody (Purified NA/LE Rat IgG1, #554682 BD Pharmingen)
- Anti-IL10 antibody (Purified NA/LE Rat Anti-Mouse IL-10, #554421)
- SiRNA oligos against Stat3, Il6ra or a nontargeting control (LifeTechnologies)
- Not explicitly mentioned
- Bone marrow-derived macrophages (BMDM)
- IL-6 (50ng/ml)
- IL-10 (10ng/ml)
- Isotype control antibody (10 μg/ml)
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