Histopaque 1077 cell separation medium

Peripheral blood was collected in EDTA-buffered collection tubes, and peripheral blood mononuclear cells (PBMCs) were isolated using Histopaque—1077 cell separation medium (Sigma Aldrich) and Leucosep tubes (Greiner Bio-one), according to manufacturer’s instructions. Lymphoblastoid cell lines (LCLs) were established by infecting B-cells from the PBMCs with Epstein Barr Virus (EBV). Briefly, PBMCs were incubated with 2 mL of culture supernatant from B95.8 cells (marmoset cell line) expressing EBV, at 37 °C in a 5% CO₂ atmosphere for 2.5 h. Then, cells were kept in RPMI 1640 (Roswell Park Memorial Institute) culture medium (Biowest), supplemented with 10% Fetal bovine serum (FBS) (Sigma Aldrich) and 1% penicillin-streptomycin (Thermo Fisher Scientific), containing phytohemagglutinin (PHA) (Sigma Aldrich) to induce the secretion of T-cell-assisted B-cell growth factors and the removal of T-cells^{86 (link),87 (link)}. One week after infection, clusters were visible, and PHA was withdrawn. For experiments, LCLs were seeded at a concentration of 3 × 10⁵ cells/mL and passaged at intervals of three days. Cells were kept in culture in a saturated, humidified atmosphere at 37 °C and 5% CO₂ for a maximum of 10 passages.

Human PBMCs were isolated by gradient density centrifugation using Histopaque-1077 cell separation medium (Sigma-Aldrich) as previously described [20 (link)]. Briefly, 1 × 10⁵ human cells were plated on 96-well plates on glass coverslips cultured for 24 hours in the presence of M-CSF (25 ng/ml), and then adhered cells were transferred to 24-well plates where they were cultured with either M-CSF (25 ng/ml), M-CSF (25 ng/ml) + RANKL (30 ng/ml) or M-CSF (25 ng/ml) + LTB4 (10 nM) for up to 14 days. Multinucleated (three or more nuclei), TRAP⁺ cells capable of F-actin ring formation, were characterized as osteoclasts. The cells cultured on plastic dishes were stained for TRAP using a commercially available kit (Sigma-Aldrich) according to the manufacturer’s instructions. F-actin ring formation was visualized using phalloidin-fluorescein isothiocyanate (FITC) staining (Sigma-Aldrich). Culture medium was collected and frozen at −80°C until EIA analysis.