Osteoclast Migration Assay

Using a transwell assay, migration of osteoclasts was evaluated as described previously with minor modifications [41 (link)]. After isolating them from vehicle control and RANKL-treated mice, bone marrow-derived cells (2 × 10⁶/ml) were cultured in M-CSF and RANKL in 6-well plates for 4 days, and then trypsinized in Hank’s balanced salt solution. With and without salubrinal (2 μM), the osteoclast precursor cells (1 × 10⁵ cells/well) were loaded onto the upper chamber of transwells and allowed to migrate to the bottom chamber through an 8-μm polycarbonate filter coated with vitronectin (Takara Bio Inc., Otsu, Shigma, Japan). α-MEM consisting of 1% bovine serum albumin (BSA) and 30 ng/ml of M-CSF was in the bottom chamber. After reacting for 6 h, the osteoclast precursor cells in the lower chamber was stained with crystal violet and counted.