Human PBMC isolation and macrophage polarization

Human PBMCs were obtained from healthy donors by density gradient centrifugation. Briefly, blood was diluted two to four times in cold PBS supplemented with 2 mM EDTA and layered on top of Ficoll-Paque (GE Healthcare) at 3:1 ratio. The mix was centrifuged (400g, 40 min, room temperature) in a swinging-bucket rotor without brake. The mononuclear cell layer ring was carefully transferred to a new tube and washed repeatedly in PBS-EDTA. After pellet resuspension, cells were labeled using a human Pan Monocyte Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions and then applied to an LS selection column attached to MidiMACS separator (Miltenyi Biotec). The eluted fraction was collected from the column, and the cells were then seeded. Macrophages were differentiated for 7 days by adding recombinant human GM-CSF (50 ng/ml) and human M-CSF (100 ng/ml) to the medium to promote M1 and M2 polarization, respectively. At day 6 after seeding, M1 cultures were then supplemented with human IFN-γ (100 ng/ml), whereas at day 5 after seeding, M2 cultures were supplemented with human IL-4 (20 ng/ml). All recombinant human cytokines were from PeproTech. Conditioned medium was collected from the last 24 hours of macrophage culture and diluted 1:1 with fresh medium before addition to cancer cell cultures.