Multi photon confocal microscope

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 see Supplementary Methods for additional details. The percentages of PMD-affected cells and dead cells were quantified (Bioquant).

MLO-Y4 cells, courtesy of Dr. Lynda Bonewald, were maintained in growth medium (α-MEM Invitrogen) + 5% fetal bovine serum (FBS, Atlanta Biologicals) + 5% bovine calf serum (HyClone) + 1% Penicillin/Streptomycin). Cells were seeded onto type 1 collagen-coated glass coverslips and cultured for 24 h in either normal culture medium or culture medium supplemented with Vitamin E (220 μM alpha-tocopherol, Sigma #T3251), or Trolox (220 μM, Sigma #238813; a water soluble form of Vitamin E). Cells were treated with vehicle or H₂O₂ (1 mM) for 10 minutes and then subjected to mechanical wounding with glass beads in the presence of lysine-fixable fluorescein-conjugated dextran (10 kDa, 5 mg/ml + 10 mg/ml BSA), as we previously described [18 (link)]. Five minutes after wounding, cells were stained with propidium iodide (0.3 mg/ml) to detect dead cells (i.e., unrepaired PMD). Images were collected at randomized positions on the coverslip, where at least 10 cells were visible per field of view, using a multi-photon confocal microscope (Zeiss). The ratio of dead to repaired cells was calculated with image analysis software (Bioquant OSTEO). Three independent trials of each experimental condition were completed.

MLO-Y4 cells were seeded 72 hours prior to experiments onto type 1 collagen-coated slides (18 (link)); cells were 70% confluent at the time of experiments. For studies utilizing reduced serum (1% FBS, 1% BCS, 1 mg/mL bovine serum albumin), cells were switched to lower-serum medium 24 hours prior to experiments. Media with fluorescein-conjugated dextran (10 kDa, 1 mg/mL) was applied to control cells (slides in static dishes) or used to apply fluid shear stresses (10 to 30 dynes/cm²) via laminar flow in a parallel plate flow chamber (Streamer, Flexcell) for as few as 7 minutes or as long as 2 hours. After each experiment, slides were rinsed in phosphate buffered saline (PBS); cells that repair PMD will retain the dye after washing (19 (link)). Slides were imaged on a multi-photon confocal microscope (Zeiss); please see Supplementary Methods for additional details. The percentage of PMD-affected cells was quantified with image analysis software (Bioquant Osteo).