Dcn228

FaDu and monocytes–FaDu spheroids were dissociated as described above, and 5 × 10⁴ cells were centrifuged for 5 min at 1200 rpm and washed with DPBS/FBS 5%. Staining for the surface marker CD206–APC (anti-human, monoclonal recombinant IgG1, DCN228, Miltenyi Biotec, Leiden, The Netherlands) was performed for 10 min at 4 °C in the dark at a 1/50 dilution in DPBS/FBS 5%. After washing, the cells were resuspended in DPBS/FBS 5% and analyzed by flow cytometry. The samples were assessed in Fluorescence-Activated Cell Sorting (Beckman Coulter NaviosTM) and the data were analyzed using Kaluza software version 2.1

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.