Lps o111 b4

BMDMs were differentiated in DMEM (Invitrogen) with 10% v/v FCS (Thermo Fisher Scientific), 10% MCSF (L929 cell supernatant), 10 mM HEPES (Invitrogen), and nonessential amino acids (Invitrogen). 1 day before infection, macrophages were seeded into 6-, 24-, or 96-well plates at a density of 1.25×10⁶, 2.5×10⁵, or 5×10⁴ per well. If required macrophages were pre-stimulated with PAM3CSK4, LPS O111:B4 (InvivoGen), mIFN-β or mIFN-γ (eBioscience). For infections with F. novicida, bacteria were grown overnight in BHI or TSB at 37 °C with aeration. The bacteria were added to the macrophages at multiplicity of infection (MOI) of 100, or as otherwise indicated. The plates were centrifuged for 15 min at 500 g to ensure comparable adhesion of the bacteria to the cells and placed at 37 °C for 120 min. Next, cells were washed and fresh medium with 10 μg/ml gentamycin (Invitrogen) was added to kill extracellular bacteria and plates were incubated for the desired length of time. Transfection with poly(dA:dT) or poly(dG:dC) was done as described previously^{29 (link)} or as indicated.