Rea968

Briefly, after polarization in T75, M1 and M2 were collected and stained for surface markers during 10 min at 4 °C in DPBS/FBS 5% using CD86–APC (anti-human, monoclonal recombinant IgG1, REA968, Miltenyi Biotec, Leiden, The Netherlands); CD163–APC (anti-human, monoclonal recombinant IgG1, REA812, Miltenyi Biotec, Leiden, The Netherlands); and CD206–APC (anti-human, monoclonal recombinant IgG1, DCN228, Miltenyi Biotec, Leiden, The Netherlands) at a dilution of 1/50. For intracellular staining, cells were first fixed and permeabilized for 20 min at 4 °C with Cytofix/Cytoperm (BD Biosciences, Erembodegem, Belgium) and then washed with PermWash buffer (BD Biosciences, Erembodegem, Belgium). Next, cells were stained for 10 min at room temperature with CD68–PE antibody (anti-human, monoclonal recombinant IgG1, REA886, Miltenyi Biotec, Leiden, The Netherlands) diluted 50× in PermWash buffer. After washing, the cells were resuspended in DPBS/FBS 5% and analyzed by flow cytometry. The samples were assessed in BD LSRFortessa X-20 and the data was analyzed using FlowJo_v10.7.1.