Estimation of parameters characterizing the dynamics of monocytes and osteoclasts.

<p>RAW 264.7 cells were plated at the density of 2.5×10³ cells/cm² (triangles), 5×10³ cells/cm² (squares), 10×10³ cells/cm² (circles) and cultured either untreated (open symbols) or in the presence of RANKL (50 ng/ml, closed symbols). A–D) At indicated times the monocytes were collected from parallel samples and numbers of live and dead cells were counted. A) Changes in monocyte number with time are similar in RANKL treated and untreated cultures. B) Linear dependence of ln(monocyte number) on time indicates first order exponential dynamics for monocyte proliferation. C) The monocytes collected at indicated days from parallel samples, were re-plated on new wells at a density of 5×10³ cells/cm² and treated with RANKL (black bars) or cultured without RANKL (open bar) for additional 5 days, when the samples were fixed and the numbers of TRAP-positive multinucleated osteoclasts were assessed. Data are mean±SEM, n = 5 independent experiments. D) Numbers of trypan blue positive (dead) monocytes were assessed at each time point and presented as a percentage of total number of monocytes. Data are mean±SD, n = 3 replicates. E) RAW 264.7 cells were plated at the indicated density and cultured in the presence of RANKL (50 ng/ml) for 5 days, when the samples were fixed and the numbers of TRAP-positive multinucleated osteoclasts were assessed. Data are means of 3 replicates for all densities except 2.5×10³, 5×10³, and 10×10³ cells/cm², when data are mean±SEM, n = 9 independent experiments. F) In 3 independent experiments the number of nuclei per osteoclast was assessed in ∼100 osteoclasts per experiment. The data are percentage of osteoclast containing certain number of nuclei from the total of 315 osteoclasts. G) The rate constant of osteoclast death was estimated form the linear dependence of ln(osteoclast number) on time, with day 0 representing the day when maximum of osteoclasts was formed in each experiment. H) During 3 independent experiments, the medium was collected at the indicated day in the end of two-day culture period. RAW 264.7 cells were plated at the density of 5×10³ cells/cm² and treated with RANKL (50 ng/ml) either without further addition (control) or supplemented with 10% osteoclast conditioned medium collected on indicated day. On day 5 the samples were fixed and the numbers of TRAP-positive multinucleated osteoclasts were assessed. Data are mean±SEM, n = 4 independent experiments, p<0.05, assessed by student t-test.