Monocyte-derived Macrophage-like Cells Generation

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of HIV seronegative donors, and monocytes prepared as described above. MMG were generated from isolated monocytes using methods modified from those previously described [34 (link), 37 (link)]. To induce the differentiation of MMG, monocytes were cultured in RPMI-1640 supplemented with 1X GlutaMAX (Life Technologies), 1% penicillin/streptomycin and a mixture of the following human recombinant cytokines: M-CSF (10 ng/mL; Peprotech), GM-CSF (10 ng/mL; Peprotech), NGF-β (10 ng/mL; Peprotech), CCL2 (100 ng/mL; Peprotech), and IL-34 (100 ng/mL; Peprotech) under standard humidified culture conditions (37 °C, 5% CO₂). The monocytes were incubated with 1% serum for the first 3 days, and thereafter, every third day, cells were supplemented with fresh serum-free media containing M-CSF, GM-CSF, NGF-β, IL-34 and CCL2. Cells were cultured for 14 days. Characterization experiments were performed on day 14.