Confocal Microscopy
The aged primary astrocytes were seeded in the optical bottom 96 well plate (Thermo-165305). Cells were treated with crocetin (25 µM) for 15 days, the media were changed every 4th day until the 15th day of treatment. Fresh media containing mitoTraker Red CMXRos (Invitrogen-M7512) and bis-benzamide was added to the cells after 15 days of treatment. After 30 min of incubation, images were captured using CQ1(Yokogawa) confocal quantitative Image Cytometer. Fifteen fields were captured from each treatment group at 40X magnification. More than 1500 cells from each group were taken for further analysis. The images were analyzed using cell pathfinder software.