HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; GE Healthcare), 1 mM sodium pyruvate, and penicillin and streptomycin (100 μg/ml) (Thermo Fisher Scientific). HeLa cells stably expressing Str-KDEL as a hook and either SBP-EGFP-CCR5 or TNF-SBP-EGFP as a reporter were obtained by transduction with lentiviral particles produced in human embryonic kidney (HEK) 293T. A clonal population was then selected using puromycin resistance and limiting dilution.

Human primary macrophages were isolated from the blood of healthy donors (Etablissement Français du Sang Ile-de-France, Site Trinité, #15/EFS/012) by density gradient sedimentation in Ficoll (GE Healthcare), followed by negative selection on magnetic beads (catalog no. 19059, Stem Cells) and adhesion on plastic at 37°C for 2 hours. Cells were then cultured in the presence of complete culture medium [RPMI 1640 supplemented with 10% FCS (Eurobio), streptomycin/penicillin (100 μg/ml), and 2 mM l-glutamine (Invitrogen/Gibco)] containing recombinant human macrophage colony-stimulating factor (rhM-CSF) (10 ng/ml) (R&D Systems) (40) for 4 to 5 days.

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