Figure 5. Effect of MK conditioned media on OC formation. (A–E) Conditioned medium (CM) was prepared from MKs isolated from young and aged, female and male mice. (A–C) Unsorted BMMs from mice (6-10 weeks) were cultured in media containing 25% (v/v) MK CM plus RANKL (80 ng/ml) and M-CSF (20 ng/ml). The control media was prepared without MKs. (A) Micrographs showing mature OCs. Scale bar is 50 μm. (B, C) The number of TRAP+ multinucleated OC (>3 nuclei) was quantified. The data show that MK CM from young female mice inhibits OC formation compared to control and aged female MK CM. The data are presented as mean ± SD of three experiments (****p<0.0001 compared to the control group). No significant difference in OC formation was observed between male groups. (D, E) Unsorted BMMs from mice (6-10 weeks) were differentiated into mature OCs in media containing 25% MK CM plus normal or reduced RANKL (80 or 26.6 ng/ml, respectively) and M-CSF (6.6 or 20 ng/ml, respectively). Mature OCs were fixed and TRAP+ OCs were counted. (F) Comparison of OCs formed in control media (no MK CM) with normal or reduced RANKL and M-CSF. Three independent experiments showed similar results and a representative figure is shown. The data are presented as mean ± SD (**p<0.01, ***p<0.001, ****p<0.0001; N= 3/group). P-values were calculated by one-way ANOVA followed by Tukey’s post-hoc test.