Osteoclastogenesis and bone resorptive capacity is impaired in splenocyte-dervied cells from macFoxp1tg mice. (A) Splenocytes, obtained by homogenization of whole mouse spleen, were incubated in 96-well plates with RANKL (200 ng/mL) and M-CSF (30 ng/mL) for up to 9 days to induce osteoclast differentiation. Osteoclasts were identified by TRAP-positive staining (original magnification 40×/0.65). (B) Total osteoclast number/well was quantified and expressed as mean plus or minus SD from splenocytes isolated from 5 individual spleens per genotype. (C) Osteolytic activity (mean ± SD) of splenocyte-differentiated osteoclasts was investigated using the OsteoLyse Assay Kit (Cambrex, East Rutherford, NJ). (D) Semiquantitative PCR of unstimulated splenocytes and splenocytes stimulated with RANKL and M-CSF for 7 days (2 individual spleens per genotype). (E) Photomicrograph of distal femur (original magnification 20×/0.40) stained for TRAP. (F) TRAP-positive area was quantified by computer-assisted imaging analysis and expressed as mean plus or minus SD from 8 individual femurs per genotype.