Differentiation and Harvesting of Human Monocyte-Derived Macrophages

CD14⁺ monocytes were harvested via apheresis from healthy volunteers as described in Moore et al.[12] (link). Monocytes were frozen in liquid nitrogen in cryopreservation solution. For SPION-labeling experiments, high-density monocyte aliquots were defrosted in a water bath, washed, and resuspended in Iscove's Modified Eagle Medium supplemented with 10% fetal bovine serum and 100 ng/mL human recombinant mCSF (Miltenyi) in low-attachment flasks (Corning) at 2 × 10⁶ monocytes/mL for 7 days, replacing mCSF every second day. After 7 days, hMDMs were harvested as described, counted and incubated overnight in 96-well plates (4 × 10⁴/well) before viability studies were performed.