Double fluorescent vital assay of phagocytosis by cultured retinal pigment epithelial cells

Purpose. The goal of this study was to develop the first vital assay system for in vitro analysis of phagocytosis of rod outer segments (ROS) by normal retinal pigment epithelial (RPE) cells and for study of the phagocytic defect in RPE of the Royal College of Surgeons (RCS) rat with inherited retinal degeneration. Required features included ability to directly visualize and quantitate the phagocytic process in living RPE cultures, and capability for subsequent quantitative analysis after fixation of the cells at any chosen time after incubation with ROS. Methods. A double fluorescent method was designed, based on the process of phagosome-lysosome fusion. For vital staining of lysosomes, confluent cultures of rat RPE cells were incubated with sulforhodamine (SR), a red fluorescent lysosomotropic dye. SR- stained cultures were challenged with isolated rat ROS tagged with fluorescein isothiocyanate (FITC), a green fluorescent probe. Results. This method was used to observe all phases of the phagocytic process in the living cells and the kinetics of ROS binding, ingestion, and phagosome-lysosome fusion were determined. Control studies showed no differences in binding, ingestion, or digestion of unstained versus FITC-stained ROS. Additionally, the phagocytic defect in dystrophic RCS rat RPE cells was confirmed using this technique. Conclusions. This relatively simple new method is useful in that it uses inexpensive, readily available reagents, it enables real-time analysis of phagocytosis experiments, and it does not require termination of the cultures for analysis of phagocytic ability.