De complemented fbs

Monocytes were incubated with RPMI 1640 (Sigma Aldrich, # 10-040) medium containing 10% de-complemented FBS (OmegaScientific, #FB-02), 1 mM Sodium Pyruvate (Gibco Thermofisher), Penicillin-Streptomycin (15140122, 1000 U/mL) and 25ng/mL-50 ng/mL of recombinant human M-CSF (StemCell #78057-2) for six days at 37°C and 5% CO₂. Fresh media was added 48 h and four days after seeding.

Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll density gradient using Ficoll Plaque Premium (Sigma GE Healthcare, #17–544-02) or BD Vacutainer CPT Tubes (BD, #362753) as described somewhere else. PBMC were washed twice with HBSS (Gibco ThermoFisher, #14175095) containing 2% BSA, 1 mm EDTA. Monocytes were obtained by negative selection using a kit (StemCell, #19359). Monocytes were incubated with RPMI 1640 (Sigma Aldrich, #10–040) medium containing 10% de-complemented FBS (OmegaScientific, #FB-02), 1 mM Sodium Pyruvate (Gibco Thermofisher), Penicillin-Streptomycin (15140122, 1000 U/mL) and 25 ng/mL-50 ng/mL of recombinant human M-CSF (StemCell, #78057–2) for six days at 37°C and 5% CO₂. Fresh media was added 48 h and four days after seeding. The mDM were incubated for five hours with 100 ng/mL of Kdo2 lipid KLA or 250 μg/mL of MSUc in RPMI 1640 medium containing 10% de-complemented FBS, 1 mM Sodium Pyruvate, Penicillin-Streptomycin and 25ng–50 ng/mL of recombinant human M-CSF. 20 μM of JNK inhibitor SP600125 (Tocris, #1496) was added to the media one hour prior treatment with MSUc.