CC-4047 enhances development of granulocytic cells unable to perform bone resorption. (A, B) An OCL formation assay was performed in the presence of 100 μM CC-4047 or 0.1% DMSO for 3 weeks and cells were stained for CD51/61⁺ (23c6 antibody) and for TRAP⁺ multinucleated OCLs. Images were obtained using an Olympus IX70 microscope equipped with a 20 ×/0.40 numeric aperture objective lens (Olympus, Melville, NY), and were acquired through Magnafire 4.1 software (Optronics, Goleta, CA). For all micrographs, original magnification is ×20. (C) After 21 days of OCL culture, cells were detached using cell dissociation buffer (Sigma-Aldrich). Cells were stained with anti–CD45-ECD and anti–CD33-PE. Data were acquired on a Dako-Cytomation MoFlo cytometer. After an incubation period of 21 days cell survival was determined by annexin V–FITC/PI double staining. The percentage of viable cells, negative for both annexin V–FITC and PI, was determined. (D) Nonadherent healthy donor bone marrow cells (2 × 10⁵ cells/well) were seeded on whale dentin slices in 96-well multiplates in 100 μL/well in α-MEM containing 20% horse serum and cultured as described in “Osteoclastic bone resorption assays.” OCL formation on dentin slices was confirmed by TRAP staining. Resorption lacunae (arrows) were stained with hematoxylin, and the pit area was quantified by light microscopy.