Human soluble rankl

“Peripheral blood mononuclear cells were obtained from blood leukocyte preparations purchased from the New York Blood Center, by density gradient centrifugation with Lymphoprep (Stemcell Technology, Vancouver, BC, Canada) using a protocol approved by the Hospital for Special Surgery institutional review board”.^{48 (link)} “CD14⁺ monocytes were obtained from peripheral blood, using antihuman CD14 magnetic beads, as per the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA, USA). Purity of monocytes was >97%, as verified by flow cytometric analysis. CD14 ⁺ cells were plated at a density of 1 × 10⁶ cells·mL⁻¹ and cultured with 20 ng·mL⁻¹ of M-CSF (Peprotech) in alpha modified essential medium (α-MEM) (Thermo Fisher Scientific) supplemented with 10% Hyclone fetal bovine serum (GE Healthcare) and 1% L-glutamine (200 mmol·L⁻¹, Thermo Fisher Scientific) for 1 day to obtain OCPs”.^{25 (link)} OCPs then were incubated with 20 ng·mL⁻¹ of M-CSF and 40 ng·mL⁻¹ of human soluble RANKL (Peprotech) to differentiate into osteoclasts. “Medium and cytokines were replenished every 3 days. When multinucleated cells were observed, cells were fixed and stained for TRAP using the Acid Phosphatase Leukocyte diagnostic kit (Sigma Aldrich) as recommended by the manufacturer. Multinucleated (>3 nuclei), TRAP-positive osteoclasts were counted in triplicate wells”.^{48 (link)}

Recombinant human M‐CSF and human soluble RANKL were purchased from PeproTech (Rocky Hill, NJ, USA). Anti‐NFATc1 (7A6) and anti‐c‐Fos (H125) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and monoclonal antibodies against β‐actin (AC‐74) and secondary antibodies were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Antibodies against ERK, JNK, p38, IκB, and Akt were from Cell Signaling Technology (Cambridge, MA, USA) as were phospho‐specific antibodies for ERK (Thr202/Tyr204), JNK (Thr182/Tyr185), p38 (Thr180/Tyr182), IκB (Ser32) and Akt (Ser473). All other reagents were obtained from Sigma‐Aldrich.