Human rankl

Peripheral blood mononuclear cells (PBMCs) were isolated from a buffy coat (Sanquin) as described previously [26 (link)]. Buffy coats were obtained from blood donated by healthy blood donors at Sanquin Blood Supply, Amsterdam, The Netherlands. PBMCs were seeded at 5x10⁵ cells/well of 96 well plates or on bovine bone slices in DMEM containing 10% FCS, antibiotics, and control-CM, CXCL8-CM from 200 pg/ml CXCL8 treatment, CCL20-CM from 500 pg/ml CCL20 treatment, CXCL8+CCL20-CM, and TNF-α-CM (ratio DMEM:CM = 1:1 (v/v)). Twenty-five ng/ml recombinant human M-CSF (R&D Systems, Minneapolis, MN) was added to the cells from day 1 to day 3. Ten ng/ml M-CSF and 4 ng/ml human RANKL (Peprotech, London, UK) were added from day 3 to day 21. To similar cultures, 0.15 μg/ml human IL-6 antibody (Clone #6708, R&D Systems) was added and IgG isotype control (Clone #11711, R&D Systems) was used as control for IL-6 antibody. PBMCs were also cultured with DMEM containing 10% FCS, antibiotics, and either CXCL8 (200 pg/ml) or CCL20 (500 pg/ml). After 3 weeks, cells were fixed in 4% formaldehyde, and stained for tartrate-resistant acid phosphatase (TRACP; Sigma). Nuclei were visualized by 4’,6-diamidino-2-phenylindole (DAPI) staining. Osteoclastogenesis was assessed by counting the number of TRACP-positive osteoclasts containing >3 nuclei per cell on 10 pre-determined microscopic fields in the each well.

Human RANKL and M-CSF were obtained from Peprotech EC Ltd. (London, UK). Akt, phospho-Akt, p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, IκB, and phospho-IκB were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA). GSK3β, phospho-GSK3β, and Runx2 antibodies were purchased from Bioworld Technology Inc. (St. Louis. Park, MN, USA). NFATc1, c-Fos, phospho-Smad, Smad, and β-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Thawed PBMCs were plated in cell-culture dishes at 8.5 × 10⁷ cells per dish in DMEM, 10% FBS, 1% antibiotics, 5% l-Glutamine and 25 ng/ml human M-CSF. After an expansion time of 72 h, the PBMCs were incubated with PBS/EDTA (Life Technologies, Darmstadt, Germany) for 10 min, detached for counting and settled in experimental set up. For osteoclast differentiation in the experimental set-up, medium was supplemented with 25 ng/ml human M-CSF (Peprotech, Rocky Hill, USA) and 50 ng/ml human RANKL (Peprotech, Rocky Hill, USA).

Osteoclasts (OCs) were obtained as previously described [5 (link), 59 (link)]. Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coat preparations from healthy donors and separated on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). PBMC were seeded at optimal density, incubated for 2h at 37°C and then washed three times with PBS to remove non-adherent cells. Cultures were grown in RPMI supplemented with 10% FCS, human M-CSF (Peprotech, London) (30 ng/ml), and human RANKL (Peprotech) (40 ng/ml). Cells were fed every 3 days with fresh medium and differentiating factors in the absence and in the presence of increasing concentrations of NZ or ZA (25,50,100 nM). After 14 days of culture cells were used for tartrate-resistant acid phosphatase- (TRAcP) staining. To quantify the formation of TRAcP positive multinucleated cells, cell cultures were stained with Leukocyte Acid Phosphatase Kit (Sigma Diagnostics), according to manufacturer's instruction. Cells positive for TRAcP and having more than three nuclei were considered as TRAcP-positive multinucleated OCs.

All cell culture reagents, including media, antibiotics and fetal bovine serum, were purchased from Gibco BRL (Grand Island, NY, USA). Recombinant human M-CSF and human RANKL were purchased from Peprotech (London, UK). Specific antibodies against p-ERK (Tyr202/204), p-P38, p-JNK (Tyr1007/1008), p-Akt, p-CREB, p-PLCγ2 (Tyr759), p-Btk (Tyr223), NFATc1 and c-Fos were obtained from Cell Signaling Technology (Boston, MA, USA). The monoclonal β-actin antibody was purchased from Sigma (St. Louis, MO, USA), and secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

All cell culture reagents, including media, antibiotics, and fetal bovine serum (FBS), were purchased from Gibco BRL (Grand Island, NY, USA). Recombinant human M-CSF and human RANKL were purchased from Peprotech (London, UK). All antibodies were obtained from Cell Signaling Technology (Boston, MA, USA).