Isolation and Culture of Murine Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) were obtained as previously described [28 (link)]. Briefly, cells were obtained from the tibia and femur of adult C57Bl/6 mice and were incubated in Roswell Park Memorial Institute medium (RPMI) 1640 (Gibco, Cat.No. 11875085, Pasley, Scotland) supplemented with 20% (v/v) of foetal bovine serum (Dutscher, Cat.No. S1018-500, Bernolsheim, France) and 30% (v/v) of L929 conditioned medium enriched in macrophage colony stimulating factor, for 7 days at 37 °C in humidified 5% CO₂. Differentiated BMDM were detached, counted, seeded in a tissue culture plate (1.0 × 10⁶ cells/mL) overnight and finally stimulated the following day as described below.