2.6 In vitro wound healing assay

HCE cells were grown to 90% confluence in 12-well plates in DMEM/F12 medium containing 5% charcoal stripped bovine serum and antibiotics. The cells were then treated with vehicle or dexamethasone or RU486 or both in the same medium containing charcoal stripped fetal bovine serum. After treatment for 24hours, a scratch was made using a sterile 200ul yellow pipette tip in the middle of the confluent monolayer. The wells were washed with the respective treatment media to remove detached and dead cells. The wells were replaced with fresh medium containing the respective treatments. Three to five bright-field images were taken along the scratch area every thirty minutes for up to 30 hours (until the scratch wound is healed) using an incubator setup with a Zeiss LSM 710 confocal microscope using a low-magnification objective of 5X to cover a large area of the healing monolayer. Images were taken only in X and Y planes. Area of wound closure was measured using the following formula: