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. 2011 Apr 8;6(4):e18689. doi: 10.1371/journal.pone.0018689

Figure 2. In vitro differentiated osteoclasts by IL-34 and RANKL show dose-dependent bone resorption activity.

Figure 2

Mouse nonadherent bone marrow cells were cultured on bone slices for 9 days in the presence of RANKL and M-CSF or RANKL with 2.5 ng/ml, 25 ng/ml, 100 ng/ml of IL-34. The bone slices with cells were fixed and stained for TRAP, and all TRAP-positive multinucleated cells were counted and analyzed under a microscope. The cells were removed followed by WGA-lectin staining for pits. Subsequent counting of resorption pits was performed with a microscope. Representative images of TRAP staining (a) and WGA-lectin staining (b) under different culturing conditions. Representative images are shown with a maginification of 20×. Bars, 100 µm. (c). Histogram of number of osteoclast-like cells, number of pits under different culturing conditions and number of pits/osteoclast (n = 5). (d). Area of Pits was quantitated using an Olympus microscope connected to a computer and the OsteoMeasure program (version 3.21; OsteoMetrics, Atlanta, GA, USA), n = 5. One-way ANOVA analysis was performed and was followed by Turkey's and Dunnett's post-hoc test by using SPSS statistic analysis software. *: The mean difference is significant at the 0.05 level.