Nile red staining

To stain the intracellular neutral lipid, the iRPE cell cultures were treated with 2.0 μg/mL Nile red (Sigma Aldrich) for 30 min at 37°C. The cells were washed with PBS three times before microscopy observation.

Since mature iRPE culture is usually 100% confluent, the Nile red-positively stained lipid droplets were quantified using ImageJ in a fixed area of 90,000 square micrometers for the number and size of Nile red-positive signals. Fluorescent particles smaller than 0.0001 square micrometers was excluded as noise.