Fig. 4

Bone marrow mononuclear cells (BMCs) were cultured in the presence of macrophage colony-stimulating factor and soluble recombinant receptor activation of nuclear factor kappa-B ligand to promote osteoclast differentiation. a Representative images of osteoclast differentiation with recombinant C5a (rC5a) by tartrate-resistant acid phosphatase staining. b The number of multi-nuclear osteoclast in each well (n = 10). c Representative images of bone resorption in the presence of rC5a by pit formation assay. d Bone resorption area by multi-nuclear osteoclast in each well was measured by ImageJ (n = 5). e–g Quantitative reverse transcription-polymerase chain reaction analysis of NFATc1, TRAF6, and MMP-9 (n = 8). h Representative images of osteoclast differentiation of BMCs by the effect of serum derived from each group of mice in the presence of an isotype control antibody or anti-C5a antibody. i The number of multi-nuclear osteoclast in each well with or without serum derived from each group of mice (n = 10). j Representative images of bone resorption by the effect of serum derived from each group of mice in the presence of an isotype control antibody or anti-C5a antibody. k Bone resorption area by multi-nuclear osteoclast in each well was measured by ImageJ (n = 5). Statistical analyses were performed by the Student’s unpaired t test or Tukey-Kramer test (*P <0.05). Data represent the mean ± standard error of the mean. Original magnification: 40×; scale bar = 1000 μm