Human m csf

Human PBMC were maintained in RPMI 1640 (Mediatech Inc.) supplemented with 10% (v/v) heat-inactivated FBS, 2 mM L-glutamine, and 1% Pen/Strep (Invitrogen Corporation, Carlsbad, CA, USA). Cell cultures were incubated at 37 °C in a humidified 5% CO₂ incubator. To derive macrophages from PBMC, cells were cultured in RPMI as above with human M-CSF (25 ng/ml; Prospec Bio, Rehovet, Israel) for 8 to 9 days. To derive dendritic cells, PBMC were cultured in RPMI containing human GM-CSF (25 ng/ml) and IL-4 (20 ng/ml; Prospec Bio) with 50 μm 2-mercaptoethanol for 8 to 9 days [16 (link)]. To derive osteoclasts, we used a modified protocol based on expanding the monocyte/macrophage population first and then inducing osteoclast differentiation. PBMC were cultured in MEMα with human M-CSF (25 ng/ml) for 7 days followed with human RANKL (50 ng/ml) and M-CSF for an additional 14 days [17 (link)]. The stock solution of 1,25D3 (1 mM) was diluted in ethanol and subsequently diluted in culture media to a concentration of 10 nM. The human parathyroid hormone 1-34 fragment (Genscript, Piscataway, NJ, USA) was diluted in culture media to concentrations of 10⁻⁸ M and 10⁻¹¹ M for use in cell stimulation experiments.