Lipopolysaccharide e coli 055 b5

Bone marrow cells harvested from femur of C57/BL6 wild-type mice were cultured for 10 days in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-Glutamine, 25 mM HEPES, 20% heat-inactivated horse serum, 30% L929 supernatant containing macrophage-colony stimulating factor (M-CSF), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco)^{48 (link)}. Bone marrow derived macrophages (BMDM) were then harvested with cold PBS (Dulbecco), and 10⁶ and 10⁵ cells per well dispensed into 48 and 96 well plates (Corning® and Costar® Multiple Well Plates), respectively. Cells were incubated with 1 µg/mL of anti GM-CSF (Clone A7.39) or IgG2b isotype control (clone MPC-11, Bio X Cell) and stimulated with M. bovis BCG (MOI:2), M. bovis BCG-GFP (MOI:4) or LPS 100 ng/mL (Lipopolysaccharide E. coli 055:B5, Sigma-Aldrich, St Louis, MO, USA), as indicated.
For bone marrow derived dendritic cells (BMDC), bone marrow cells were cultured for 10 days in Roswell Park Memorial Institute RPMI medium supplemented with 25 mM Hepes, 1 mM sodium pyruvate, 40 µg/ml gentamycin, 50 µmol/L 2-mercaptoethanol, 10 mM L-Glutamine, 0.2% vitamins (Gibco^®), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco), 10% decomplemented FCS and 500 µL/plate of J558 supernatants containing GM-CSF^{49 (link)}. BMDC were then harvested and stimulated with M. bovis BCG (MOI:2) or 100 ng/mL LPS, as above.