Number of TRAP-positive multinucleated osteoclasts formed in the co-cultures of 5TGM1 myeloma cells and primary mouse bone marrow cells.
(A) 5TGM1 cells (1 × 103) were plated together with primary mouse bone marrow cells (BMC, 1 × 106) in 48-well plates, and cultured for 6 days. Cells were fixed and stained for TRAP activity as described in text. TRAP-positive cells with more than 3 nuclei were manually counted under a microscope as described.24,26 Data are expressed as mean ± SE (n = 6). *Significantly different from BMC alone (P < .01). (Bi) Double staining for TRAP and VCAM-1 of the co-cultures. Note that TRAP-positive large multinucleated osteoclasts (red) were surrounded by fibroblast-like VCAM-1–positive cells (black), which are most likely stromal cells. 5TGM1 myeloma cells were washed away during processing for staining. (Bii) Pit formation on dentine slices by TRAP-positive multinucleated osteoclasts formed in the co-culture. Co-culture was carried out on sperm whale dentine slices. After 6 days of culture, these dentine slices were fixed and examined by scanning electron microscopy for resorption pit formation as described.24 25
