Bz x700 digital

To test for cellular senescence, scleral cells were seeded and grown on a 12-well plate at a cell concentration of ~2500 cells per well under the same 3 treatment groups (3 wells per group), and treated daily for 7 days with DEX, MED, or DMSO. At the end of day 7, cells were washed once with PBS and a fixative solution from a senescence detection kit (ab65351; Abcam) was applied for 25 minutes at RT. Cells were washed twice with PBS, treated with X-gal staining solution, inserted into a plastic bag, and placed in an incubator (37°C; 5% CO₂) overnight. Images were taken with a BZ-X700 digital imaging microscope (Keyence) at a 10X magnification. For each well, images with individual cells were counted and identified as either normal (not blue) or senescent (blue) by a masked grader. The number of blue cells was then divided by the total number of counted cells yielding the percentage of senescent cells within the image. The calculated values for each image was averaged across the 3 wells per treatment and converted into a final percentage.