Figure 1. JC-1 stained mock treated (DMSO) HLE-B3 cells under several adaptations of hypoxic and atmospheric oxygen exposure. Human lens epithelial (HLE-B3) cells were stained with JC-1 (5 µg/ml) and observed with confocal microscopy with images captured every 2.5 min over a 60 min period, under the following conditions: (1) continuous hypoxia, (2) continuous atmospheric oxygen, (3) switching from hypoxia to atmospheric oxygen, or (4) switching from atmospheric oxygen to hypoxia. Top row: (continuous oxygen) HLE-B3 cells were maintained in atmospheric oxygen and stained for 30 min in atmospheric oxygen. At the end of the 30 min staining period, fresh oxygenated media without the JC-1 dye were added to the dishes. A random field of cells was then imaged. Note to the reader: The term “random field of cells” here and with all successive JC-1 analyses is meant to infer an arbitrary field of cells is selected, but once chosen, the same field of cells is photographed throughout the 60 min image capture. Second row: (atmospheric oxygen to hypoxia) HLE-B3 cells were maintained in atmospheric oxygen and stained for 30 min in atmospheric oxygen. At the end of the 30 min staining period, the cells were switched to hypoxic media (i.e., medium that had been preincubated at 1% oxygen). A random field of cells was immediately imaged. Third row: (hypoxia to atmospheric oxygen) HLE-B3 cells were placed in hypoxic conditions for 180 min. At the end of the hypoxic exposure, the media were removed and replaced with fresh oxygenated media containing JC-1. The cells were stained for 30 min in atmospheric oxygen. After this 30 min period, the media were again removed, and fresh oxygenated media were added without the JC-1 dye. A random field of cells was then imaged. Fourth row: (continuous hypoxia) HLE-B3 cells were stained with serum-free minimal essential media (MEM) cotaining JC-1 for 30 min in atmospheric oxygen. At the end of this 30 min period, the media were removed, and fresh medium that had been preincubated at 1% oxygen was added without the JC-1 dye. The cells were then switched into the hypoxic conditions for 180 min. The cells were imaged following the 3 h hypoxic exposure. Under all experimental conditions, there was no evidence of loss of membrane potential throughout the 60 min image capture.