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.