Isolation and Culture of Mouse Monocytes and Macrophages

Male C57BL/6 mice, 8–10 weeks old (Charles River Laboratories), were used in all experiments for the generation of monocytes and Mϕ. The animals were culled by CO₂ euthanasia immediately before the start of the bone marrow isolation procedure. Mouse bone marrow was harvested as previously described.³² (link) Briefly bone marrow was collected by flushing the hind-limb tibia and femur with DMEM (Gibco) containing 10% FBS (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). Cell suspension was filtered with a 70-μm cell strainer resuspended in 5 mL cold 1× red blood cell lysis buffer (BioLegend) before washing in PBS and performing gradient centrifugation using Histopaque-1077 (Sigma-Aldrich) and at 400 × g without brake for 30 min. The interphase was carefully collected as bone marrow mononuclear cells (BMMNCs). For monocyte collection, collected BMMNCs were subjected to magnetic sorting with the Monocyte Isolation Kit (Miltenyi Biotec) and MS Separation Columns (Miltenyi Biotec). For Mϕ production, isolated BMMNCs were plated at a density of 4 × 10³ cells/cm² and cultured for 6 days in complete DMEM containing mouse macrophage colony stimulating factor (M-CSF; 20 ng/mL; PeproTech) to differentiate to Mϕ.³² (link)