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Osteoclasts are large multinucleated cells responsible for the resorption of bone and other mineralized tissues during physiological remodeling and pathological bone loss.Active osteoclasts are highly motile and alternate between cycles of bone resorption and migration.However, little is known regarding the subcellular mechanisms that regulate osteoclast motility.We hypothesized that changes in the concentration of cytosolic free calcium contribute to the regulation of osteoclast migration.Our purpose was to characterize subcellular changes in cytosolic calcium of osteoclasts and the possible role of calcium in regulating osteoclast motility.Cytosolic calcium was monitored by digital fluorescence imaging of fura-2-1oaded osteoclasts using alternating excitation wavelengths of 345 and 380 nm with emission at 510 nm.The pixel-by-pixel ratio of fluorescence intensity at 345/380 nm provided a spatial map of calcium concentration within the cell.Changes in osteoclast morphology were captured by trine-lapse microscopy on an inverted phase-contrast microscope coupled to a CCD camera.