Osteoclastogenesis in vitro from sorted B cells. (A) TRAP staining of osteoclasts derived from the indicated sorted cells originating from BM (10,000 cells per well) and cultured with M-CSF and RANKL. Left - Pro-B cells expressing CD115 (B220⁺CD19⁺CD43^HighIgM^-CD115⁺). Right - Pro-B cells not expressing CD115 (B220⁺CD19⁺CD43^HighIgM^-CD115^-). Data are mean±SEM of osteoclast area, n=5 mice in each group; *p<0.05. (B) Pit resorption area from the indicated sorted cells cultured on calcium phosphate-coated 96-well plates with M-CSF and RANKL. (C) Transdifferentiation of 180,000 cells per well CD19-Cre;R26R-EYFP into osteoclasts. DRAQ5 (blue) and anti-GFP (green) for CD19-Cre;R26R-EYFP and CD19-Cre cells. Bottom - TRAP staining. Confocal images (x20 magnification) (D) EPO increased differentiation of Pro-B cells into osteoclasts in vitro. TRAP staining of osteoclasts derived from sorted B220⁺CD19⁺CD43^HighIgM^- cells (180,000 per well) utilizing same culture conditions as in A ± 5U/ml EPO, n=7 mice in each group. Data are % osteoclasts of a representative image; EPO versus Control, displayed a 70% increase, p<0.05.