Isolation and Differentiation of Monocyte-Derived Macrophages

Primary human gingival epithelial cells (pHGEs) were purchased from CELLnTEC (catalog no. HGEPp; CELLnTEC, Research Triangle Park, NC) and maintained in the fully supplemented culture medium (catalog no. CnT-PR; CELLnTEC). Freshly prepared buffy coats were isolated from healthy donors by density gradient centrifugation as described previously (19 (link), 20 (link)). Briefly, mononuclear cells from 50 ml peripheral blood were purified with Ficoll Paque PLUS (catalog no. 17-1440-03; GE Healthcare, Pittsburgh, PA)–based density centrifugation. Purified cells were then incubated with magnetic-labeled CD14 beads (catalog no. 130-050-201; Miltenyi Biotec, San Diego, CA) to isolate monocytic cells according to manufacturer’s instruction. Purified monocytic cells from healthy donors were then plated at a density of 0.25 × 10⁵ per well onto a 96-well plate and cultured in RPMI 1640 supplemented with penicillin–streptomycin and 10% FBS. For monocyte-derived macrophage differentiation, M-CSF (catalog no. 300-25-10UG; PeproTech, Rocky Hill, NJ) was added to the culture medium DMEM to achieve a final concentration of 100 ng/ml after monocytes were plated. Culture media supplemented with M-CSF was replenished every 72 h.