Osteoclast Precursor Cell Chemotaxis

RAW264.7, a murine macrophage/monocyte lineage cell line, and mouse bone marrow-derived M-CSF-dependent monocytes (BM-MDM), containing osteoclast precursor cells, were cultured and stimulated to differentiate using RANKL as previously described28 (link),29 (link). In vitro chemotactic activity was evaluated both using a modified Boyden’s chamber30 (link) or with a visually-accessible chemotactic chamber, EZ-Taxiscan. In vivo S1P-directed chemotaxis of OP monocytes was visualized by intravital two-photon microscopy of mouse calvaria bone tissues of heterozygous CX₃CR1-EGFP knock-in mice12 (link) and of CSF1R-EGFP transgenic14 (link) mice, according to a protocol modified from a previous report10 (link),11 (link). The generation of the loxP-flanked S1P₁ allele (S1P₁^loxP)18 (link) and osteoclast/monocyte lineage-specific CD11b-Cre transgenic mice19 (link) have been described previously. Histomorphometry of femurs from OC/monocyte-lineage specific S1P₁ conditional knockout (cS1P₁^-/-) mice, as well as mice ovariectomized / sham-operated and treated with FTY720 or vehicle, were performed using μCT. Assessment of osteoclast attachment to the bone surface was performed using a newly developed fully automated segmentation approach to analyze two-photon images of bone tissue sections in which OCs and bone trabeculae were visualized with fluorescent TRAP staining20 (link) and 2nd harmonic generation from collagen fibers21 (link), respectively. To examine the composition of peripheral blood mononuclear cells (PMCs), the cells were stained with fluorophore-conjugated anti-F4/80, anti-CD11b, and anti-CD3 using conventional methods. Flow cytometric data were collected on a FACSCalibur and analyzed with FlowJo software. All mice were bred and maintained under specific pathogen-free conditions at the animal facility of NIH, according to NIH institutional guidelines under an approved protocol. For statistical analyses, the Mann-Whitney rank sum test was used to calculate p values for highly skewed distributions. For Gaussian-like distributions, two-tailed t-tests were used.