Additional file 3: Figure S3. of Chemokine signals are crucial for enhanced homing and differentiation of circulating osteoclast progenitor cells

OCs differentiated from PBMCs exhibit OC-specific phenotype and bone-resorbing activity. The number of multinucleated cells expressing TRAP was used for quantification of differentiated OCs under stimulation by M-CSF and RANKL. To further confirm the identity of OCs, parallel cultures were performed and stained for VNR expression. Functional bone-resorbing activity of differentiated OCs was confirmed by pit formation assays using bovine cortical bone slices under the same culture conditions. Presented are representative images of osteoclastogenic cultures of PBMCs from control subjects and patients with RA. In addition, cultures from patients with RA stimulated with CCL5 (10 ng/ml) are shown to confirm the osteoclastogenic effect of CCL5. Red arrows indicate bone resortion pits, formed by active mature osteoclasts. TRAP-stained mature OCs and bovine cortical bone slices were imaged under light microscopy at × 200 magnification. VNR-expressing mature OCs were imaged using a fluorescence microscope at × 200 magnification. (TIF 4477 kb)