Abstract
Although it is possible to use a tartrate-resistant acid phosphatase (TRAP) stain to assist in identifying osteoclasts, a separate method is needed to determine the bone resorption activity of osteoclasts. Since osteoclasts leave “pits” after bone matrix resorption (Charles et al., 2014), it is possible to stain pits as a method of measuring osteoclast bone resorption activity. The pit assay protocol enables researchers to stain bony slices that were co-cultured with osteoclasts with toluidine blue in order to allow the visualization, capture, and analysis of osteoclast resorptive activity based on the number, size and depth of pits (Zhou et al., 2015). The pit assay protocol is separated into three sequential stages: Preparation of bone slices (1); preparation of osteoclast precursors (Ross et al., 2006; Teitelbaum et al., 2000) (2), and bone resorption pit assay (3).
Keywords: Osteoclast function, Bone resorption, Pit assay
Materials and Reagents
Equipment
Procedure
Representative data
The following three representative examples have been taken from Zhou et al. (2015). Figure 4. Illustrative examples of positive results of pit assays. The pit assay experiments shown above were conducted on dentin slices co-cultured with osteoclasts for 10 days in vitro. The pit assay results in 4A and 4B were generated without the addition of an extraneous SHP2 inhibitor. In 4C, however, SHP2 was inhibited with NSC-87877 during the co-culture process to block mature osteoclast formation (Zhou et al., 2015). Hence, a lack of mature osteoclasts was induced, which caused a lack of resorption pits as shown in 4C. Scale bar, 100 µm.
The “pits” created by the pit assay procedure are the blue areas in Figure 4A, Figure 4B, and Figure 4C. The red arrows in Figure 4A, 4B and 4C point to the pits created. The blue staining is due to the toluidine blue used in the procedure. The samples represented in Figure 4A and 4B both have a decent amount of pits. The sample represented in figure 4C has a relative absence of pits. The mice strain used in the original experiment is the C57BL/6 strain.
Recipes
Acknowledgments
This protocol was adapted from the previously published paper Zhou et al. (2015). This work was supported in part by the U. S. National Institutes of Health (NIH) National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R21AR57156 (to W. Y.) and NIH National Institute of General Medical Sciences Grant P20 GM103468. This study was also aided by a grant from the Pediatric Orthopaedic Society of North America and the Orthopaedic Research and Education Foundation (to W. Y.), and the U. S. Department of Agriculture Research Service program Grant #5450-51000- 046-00D (to J. C.).
References
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