MLO-Y4, OCY454, and primary osteocytes were wounded by glass beads, as previously described (8 (link), 19 (link)), to facilitate wounding large numbers of cells. Vitamin E (Sigma T3251, 220 μM) (19 (link)) or calpeptin (Sigma C8999, 20 μM) (20 (link)) were introduced as indicated 24 hours prior to wounding. Whole cell lysates were collected for western blotting as previously described (21 ), using antibodies against c-fos (Santa Cruz) and β-actin (Sigma). Please see Supplementary Methods for additional details.
To assess cell survival after mechanical wounding, MLO-Y4 cells were wounded by glass beads in the presence of lysine-fixable fluorescein-conjugated dextran (10 kDa, 5 mg/mL+10 mg/mL BSA) containing either 1.8 mM Ca2+, 1.8 mM Ca2++20 μM calpeptin, or 1.5 mM EGTA. Five minutes after wounding, cells were stained with propidium iodide (0.3 μg/mL) to detect dead cells (i.e., unrepaired PMD) and imaged on a multi-photon confocal microscope (Zeiss). Please seeSupplementary Methods for additional details. The percentages of PMD-affected cells and dead cells were quantified (Bioquant).
To assess cell survival after mechanical wounding, MLO-Y4 cells were wounded by glass beads in the presence of lysine-fixable fluorescein-conjugated dextran (10 kDa, 5 mg/mL+10 mg/mL BSA) containing either 1.8 mM Ca2+, 1.8 mM Ca2++20 μM calpeptin, or 1.5 mM EGTA. Five minutes after wounding, cells were stained with propidium iodide (0.3 μg/mL) to detect dead cells (i.e., unrepaired PMD) and imaged on a multi-photon confocal microscope (Zeiss). Please see
