CFDA-SE stock solutions (10 mM) were diluted to concentrations of 5, 10, 20, 40, and 80 μM with PBS. Secondary cultures of newborn rabbit RPE were harvested and washed three times with PBS. After cell viability was measured by trypan blue, 1.0 mL of 5 × 104 cells/mL PBS was added to 16 10-mL glass centrifuge tubes. Immediately, each tube received 1.0 mL of a CFDA-SE dilution to make the final concentrations of 2.5, 5, 10, 20, and 40 μM. There were three tubes for each concentration, and one tube, without CFDA-SE, served as the control. Tubes from each concentration were incubated at 37°C with shaking for 1, 5, or 10 minutes. The RPE cells were washed three times with PBS after incubation. The viability of RPE cells in each of the 16 tubes was tested by trypan blue staining, and the fluorescence was observed by fluorescence microscopy. Then, the cells were plated on laminin-coated tissue culture plates (Corning-Costar, Corning, NY) with DMEM-F12 medium supplemented with 20% FBS. To determine the plating efficiency of labeled RPE cells, the number of cells attached to the plate 24 hours after plating was divided by the number of the RPE cells added to the plate. Each experiment was performed in triplicate. Based on cell viability, plating efficiency, and fluorescence intensity, an optimal labeling condition was selected for the following in vitro experiments.