Abstract
The Bone resorption assay provides an easy to use protocol for quantitatively measuring in vitro osteoclast-mediated bone resorption. Osteoclasts can be seeded onto the bone slices and formation of resorption pits can be quantified via toluidinblue staining (Scholtysek et al., 2013).
Materials and Reagents
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Osteoclasts
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Bone slices (IDS PLC, catalog number: DT-1BON1000-96 )
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Isopropanol
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Toluidine blue O (Sigma-Aldrich, catalog number: 198161 )
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MilliQ water
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96 well plates for cell culture (Greiner Bio-One GmbH, catalog numer: 650185 )
Equipment
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OsteoMeasure System (http://www.osteometrics.com/product_info.htm)
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Ultrasonic water bath
Procedure
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Osteoclasts were plated (250,000 cells/well) in the presence and absence of calvarial osteoblasts in 96-well plates, previously equipped with bone slices.
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Cells were cultured in appropriate media for at least 14 days at 37 °C.
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After this time cells were washed twice with PBS and removed from bone slices via ultrasonication in 250 µl of 70% isopropanol for at least 15 min at high power.
Note: A volume of 250 µl of 70% isopropanol before ultrasonication makes it easier to get rid of the cells.
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Resorption pit formation was visualized by 100 µl toluidine blue (1%; dissolved in water) staining for 2 min at RT.
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The slices were then rinsed with 250 µl MilliQ water at least 5 times to wash out residues.
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Resorption pits are now stained in dark blue (Figure 1) and resorption area can be quantified via Osteomeasure System or resorption pits/well can be counted via light microscopy.

Figure 1. Resorption pit formation
Acknowledgments
This protocol was adapted from the previously published paper Scholtysek et al. (2013).
References
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Scholtysek, C., Katzenbeisser, J., Fu, H., Uderhardt, S., Ipseiz, N., Stoll, C., Zaiss, M. M., Stock, M., Donhauser, L., Bohm, C., Kleyer, A., Hess, A., Engelke, K., David, J. P., Djouad, F., Tuckermann, J. P., Desvergne, B., Schett, G. and Krönke, G. (2013). PPARbeta/delta governs Wnt signaling and bone turnover. Nat Med 19(5): 608-613.
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Scholtysek, C., Krönke, G. and Schett, G. (2014). Bone Resorption Assay.
Bio-protocol 4(14): e1187. DOI:
10.21769/BioProtoc.1187.