C57Bl/6 mice were purchased from JAX Mice and Services, Bar Harbor, ME, USA. The mice were handled in accordance with protocol approved by the Institutional Animal Care and Use Committee of the University of Rochester (Rochester, NY, USA).
Isolation and culture of cells to generate OC
Mouse bone marrow cells were obtained aseptically as previously described (Takeshita et al., 2000
), by flushing the femora and tibiae of 20-week-old C57BL/6 mice with sterile 1X phosphate-buffered saline (PBS). Red blood cells in the collected suspension were lysed with ammonium chloride solution (Stem Cell Technologies), and the collected cells were cultured in minimal essential media- alpha modification (alpha-MEM) (Invitrogen, Carlsbad, CA, USA), supplemented with 10% heat-inactivated fetal calf serum, 5% penicillin / streptomycin, and 5% minimal essential medium nonessential amino acids (Invitrogen) with a final pH of 7.4. The cells were incubated at 37 °C in 5% CO2
atmosphere. M-CSF (50 ng/mL) was added to the bone marrow-derived cells for 3 days to enrich the adherent CD11b+ (Mac-1+) population as previously described (Hayashi et al., 2002
). After 3 days, 100 ng/mL RANKL (Cell Sciences, Canton, MA) in alpha-MEM was added to the cells for 3 more days to generate OC. RAW 264.7 cells or RAW 264.7 GFP cells were treated with 100 ng/mL RANKL in alpha-MEM for 3 days to generate OC.
Total RNA was extracted from the cells using the Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA) and subsequently reverse-transcribed into cDNA using the iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Real-time PCR reactions were performed in triplicate using the Rotor-Gene 3000 thermal cycler (Corbett Life Science, Sydney, NSW, Australia). The RT2
qPCR Primer Assay for mouse Dc-stamp
(Superarray, Frederick, MD, USA) was used for PCR analysis. As in previous reports (Yagi et al., 2007
served as the internal house-keeping gene control (forward primer sequence 5′-AGATGTGGATCAGCAAGCAG-3′, reverse primer sequence 5′-GCGCAAGTTAGGTTTTGTCA-3). SYBR Green I (Applied Biosystems, Foster City, CA, USA) was used for detection of DNA synthesis under the following cycling conditions: 95 °C, 10 min followed by 45 cycles of 95 °C, 15 sec; 60 °C, 60 sec; 72 °C 15 sec. The amount of Dc-stamp
mRNA expressed was normalized to the β-actin
expression for the specific treatment condition.
For OC markers and fusion-related genes, the following table depicts the various primers used and their annealing temperatures:
(F: forward sequence, R: reverse sequence)
A monoclonal antibody against DC-STAMP was generated by immunizing mice with a fifteen amino-acid peptide sharing homology to a sequence in the fourth extracellular domain of both murine and human DC-STAMP. Hybridoma clones with strong signals by EIA generated by this immunization procedure were expanded in SAFC EX-CELL 610 HSF serum-free media (Sigma, St. Louis, MO, USA) and used to generate antibody, which was purified using HiTrap protein G and PD10 columns (GE Healthcare Biosciences, Piscataway, NJ, USA). The 1A2 clone from this process was evaluated and used for all analyses in this study. Conjugation to FITC was performed using the Molecular Probes labeling kit (Invitrogen).
Immunoprecipitation-western blot (IP-western)
The anti-DC-STAMP monoclonal antibody (mAb) was used to immunoprecipitate DC-STAMP from cells treated with RANKL for 2 days. The Native Membrane Protein Extraction Kit (Calbiochem, San Diego, CA, USA) was used to extract membrane fraction proteins according to the manufacturer’s instructions. The extracted membrane fraction proteins were immunoprecipitated using the EZView Red Protein A Affinity Gel beads (Sigma) after incubation for 1 hour at 4 °C with anti-DC-STAMP mAb to form antibody-antigen complexes.
Following immunoprecipitation, immunoblotting was done using by loading the immunoprecipitated protein onto a 10% gel for SDS-PAGE. The separated proteins were blotted onto a PVDF membrane using a wet-transfer method. After transfer, the membrane was blocked for 1 hr at room temperature with 5% BSA (Sigma) in TBST. Then, either the anti-DC-STAMP mAb clone 1A2, commercially-available rabbit anti-mouse DC-STAMP polyclonal antibody KR104 (Cosmo Bio, Tokyo, Japan), or mouse IgG (Caltag, Burlingame, CA, USA) was added at a 1:1000 dilution in the blocking solution overnight at 4 °C. Following washes in TBST, goat anti-mouse IgG horse-radish peroxidase conjugate (BioRad) was added at a 1:3000 dilution, followed by more TBST washes. The blots were developed with the SuperSignal West Femto chemiluminescent substrate kit (Pierce, Rockford, IL, USA) and imaged on Kodak scientific films (Eastman Kodak, Rochester, NY, USA).
RAW cells and murine bone marrow macrophages were stained in the dark on ice with FITC-conjugated anti-DC-STAMP mAb for 30 min in 1X PBS containing 4% FCS after adding anti- CD16/CD32 to block Fc-receptors (BD Pharmingen, San Jose, CA, USA) for 15 min. 7AAD (Invitrogen) was also added to cells during the 30 min incubation, and it was used to determine which cells to gate for subsequent analysis of DC-STAMP surface protein expression. Permeabilization of the cells for intracellular staining was performed using the Cytofix/Cytoperm kit (BD Biosciences Pharmingen). If cells could not be analyzed the same day, they were fixed in 1% PFA and analyzed the following day. Flow cytometry was performed using a FACSCalibur (Becton Dickinson), FACS was performed on the same day of staining using either a FACS Vantage (Becton Dickinson) and data analysis was done with WinMDI 2.9 software (Scripps Research Institute, La Jolla, CA, USA).
Murine bone marrow macrophages cultured on glass coverslips in 12-well dishes with 100 ng/mL RANKL. Cells were fixed in 4% PFA for 20 min at room temperature for immunofluorescent staining. The cells were then blocked and permeabilized with PBS containing 0.2% BSA and 0.1% saponin for 15 min. The coverslips were incubated at room temperature for 2 hr in a humid chamber with 1A2 anti-DC-STAMP mAb. After 2 hr, PE secondary antibody, DAPI, or phalloidin was added for 45 min at room temperature in the blocking and permeabilization solution. Following the incubation, the coverslips were washed with PBS and mounted on slides for imaging. Images were then assembled, pseudo-colored, and overlaid using Adobe Photoshop 7.0 software (Adobe Systems, San Jose, CA, USA).
Data are presented as means ± standard error of the mean. Student’s t-test or analysis of variance (ANOVA) were performed with a significance level of P<0.05. Statistics were calculated using either Microsoft Excel 9.0 software (Microsoft, Redmond, WA, USA) or the GraphPad PRISM software package (GraphPad Software, La Jolla, CA, USA).