Osteoblast, osteoclast and macrophage differentiation. (A) MC3T3-E1 cells were seeded in 24-well culture plates and induced to differentiate into osteoblasts via the addition of medium containing 50 μg/ml ascorbic acid and 10 mM β-glycerophosphate. Cells were harvested at various time points throughout differentiation for gene expression studies. The figure represents MC3T3-E1 cells at various stages during the developmental process. The cells were stained with Alizarin Red S which is retained by mineralized extracellular matrix. (B) Bone marrow derived monocytes were seeded in 24-well culture plates and induced to differentiate into macrophages or osteoclasts via the addition of M-CSF or M-CSF + RANKL respectively. The figure displays representative photos of the macrophages and osteoclasts used in the study. The cells were stained with rhodamine phalloidin (F-actin stain) and DAPI (nucleic acid stain).