Isolation and Differentiation of Mouse Monocytes and Dendritic Cells

Bone marrow cells were isolated from the femurs of C57BL/6 mice in cold MAC buffer (Ca²⁺, Mg²⁺ free PBS + 2 mM EDTA + 0.5% BSA), centrifuged at 1,200 rpm for 10 min, resuspended in 5 mL RBC Lysis Buffer (1×, BD Pharm Lyse) and incubated for 5 min at RT. Reaction was terminated in PBS, and cells were centrifuged at 1,200 rpm for 10 min at RT. Cells were resuspended in 5 mL of MAC buffer and carefully added in the top of 5 mL of Histopaque solution (Sigma-Aldrich) in 15 mL tubes and centrifuged at 1,200 rpm, 25 min at 15°C without brake and one acceleration. The monocyte-enriched fraction was collected in a new tube and washed in PBS. Monocytes were further incubated with M-CSF-1 (10 ng/mL) in complete DMEM media (Thermo Fisher) to generate MOs (13 (link)), or GM-CSF (50 ng/mL) plus IL-4 (25 ng/mL) in complete RPMI to generate myeloid DCs (17 (link), 33 (link)). To generate macrophage-conditioned media (MCM) for the experiment described in Figure 6, MOs were incubated with TCM, MIF (200 ng/mL) or left untreated, in the presence or absence of C36L1 peptide (200 µM) for 72 h, and further incubated in serum-free medium for 48 h. Then, the medium was harvested, centrifuged, and filtered for functional assays or stored at −20°C.