Osteoclast differentiation assay and morphologic TRAPase staining

Bone marrow cells were flashed out from femurs of mice from the second sac groups. They were suspended in the six‐well plates for 48 h, then floating nonadherent cells were collected, whereas attached stromal cells were kept until 80% confluent to isolate RNA for real‐time PCR analysis described above. Half of the hematopoietic nonadherent bone marrow cells were used for isolation of RNA for real‐time PCR experiment as described above. Another half of the hematopoietic nonadherent bone marrow cells were cultured in 96‐well plates (2 × 10⁴ cells/well) in the presence of 30 ng/ml of RANKL. As we described previously,^{( 37 )} after 5 days for nonadherent bone marrow cell cultures, the cells were fixed with 4% paraformaldehyde and stained for TRAPase activity using a TRAPase staining kit according to the manufacturer's protocols (acid phosphatase leukocyte, procedure No. 386; Sigma‐Aldrich). TRAP‐positive cells containing more than three nuclei in each well were counted as osteoclasts under an epifluorescent microscope (model BH‐2; Olympus, Imaging America Inc).