Effects of salubrinal on migration and adhesion of pre-osteoclasts. Bone marrow-derived cells (2 × 106/ml) were cultured in M-CSF and RANKL in 6-well plates for 4 days to obtain pre-osteoclasts used for the migration and adhesion assays. A: Number of migratory cells. Osteoclast precursor cells (1 × 105 cells/well) were loaded onto the upper chamber of transwells in the presence and absence of 2 μM salubrinal. The bottom chamber was filled with α-MEM consisting of 1% BSA and 30 ng/ml of M-CSF, and cells were allowed to migrate to the bottom chamber through an 8-μm polycarbonate filter coated with vitronectin. After reacting for 6 h, the cells in the lower chamber was stained with crystal violet and counted. The images display 2 pairs of osteoclast cultures. Bar = 200 μm. B: Number of adherent cells. Ninety-six well plates were coated with 5 μg/ml vitronectin and filled with α-MEM supplemented with 30 ng/ml M-CSF. Approximately 1 × 105 osteoclast precursor cells were cultured per well in the presence and absence of 2 μM salubrinal for 30 min. Cells were stained with crystal violet and the number of cells adherent to αvβ3 integrin was counted. Bar = 200 μm.