Phagocytosis assay in vitro

To generate M1 or M2 macrophages, sorted BM cells were treated with recombinant mouse macrophage colony-stimulating factor (M-CSF). M1 polarization was achieved with further treatment through interferon gamma (IFN-γ) stimulation, followed by lipopolysaccharide (LPS). M2 polarization was obtained by further treatment with IL-4 and IL-13 [21].

Tumor cells were labeled with CFSE and co-incubated with murine BM-derived differentially polarized macrophages. Two different primary tumor cell lines (BBN1617 and MB49) were offered to either mouse M1 or M2 macrophages as targets. After the phagocytosis assays finished, staining of macrophages and the respective antibody master mix was done directly. The macrophage population was identified by macrophage markers CD11b and F4/80 targeted fluorescently labeled antibodies. Macrophages for each macrophage subtype was quantified by the percentage of CFSE+ events among CD206+ (mouse M2 macrophage), CD80+ (mouse M1 macrophages) events.