Tibia and femur from 6 to 8 week-old C57BL/6J and genetically-matched tlr2 2/2 , trif 2/2 mice were collected in ice cold PBS. Bones were sterilized with 70% ethanol and flushed with a 25-G needle using cold DMEM supplemented with 10% FCS, 10% LCM and 1% penicillin-streptomycin. Cells were seeded onto 6-well plates (Nunc) at a concentration of 10 6 cells per well and incubated at 37uC with 5% CO 2 . After 4 days, complete medium was added and cells were split at a ratio of 1:2. After 8 days, macrophages were fully differentiated.