Figure 4. Evaluation of the circulating or BM-resident OCP.

A. Flow cytometric analysis of cells from the blood of wild type (WT) and TRAP tdTomato mice before parabiosis and 2 and 4 weeks after establishing parabiosis. Live, nucleated blood cells were analyzed for expression of CD45.1 and CD45.2. The percentages of cells expressing only CD45.1 or CD45.2 are shown. Experiments were repeated eight times with similar results and representative samples are presented.

B. Flow cytometric analysis of cells from the spleen of wild type (WT) and TRAP tdTomato mice after 4 weeks of parabiosis.

C. Flow cytometric analysis of cells from the bone marrow of wild type (WT) and TRAP tdTomato mice after 4 weeks of parabiosis.

Live, nucleated blood cells were analyzed for expression of CD45.1 and CD45.2. The percentages of cells expressing only CD45.1 or CD45.2 are shown. Experiments were repeated eight times with similar results and representative samples are shown.

D. Upper Graph: Analysis of CD45.1 cells from the wild type mice and CD45.2 cells from the TRAP tdTomato mice in the bone marrow of each parabiont calculated as the percent of total nucleated cells.

Lower Graph: The number of fluorescent osteoclasts per well that were present in BMM cultures from the wild type and TRAP tdTomato parabionts after 4 weeks. Cells were cultured with M-CSF + RANKL (30 ng/ml for each) for 5 days and then examined under UV light for the number of multinucleated fluorescence cells/well. N=4 for all groups. Experiments were repeated twice with similar results. Data are presented as mean ± SEM. * Significantly different p<0.01.

E. Representative phase contrast and epifluorescent images of cultured bone marrow cells (after 6 days of culture) and bone marrow macrophages (after 5 days of culture) from wild type (WT) and TRAP tdTomato parabiont after 4 weeks. Cells were cultured with M-CSF + RANKL (30 ng/ml for each) and imaged under fluorescent light to demonstrate tdTomato (red) expression. Experiments were repeated eight times with similar results and representative samples are shown. Scale bar, 200 μm. * Significantly different from the other parabiont, p<0.01