A SIMPLE FLOW CYTOMETRIC TECHNIQUE TO QUANTIFY ROD OUTER SEGMENT PHAGOCYTOSIS IN CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS

Purpose. The primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS ) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A s econdary aim was to compare the kinetics of ROS phagocytosis in an imm ortal human RPE cell line with untransformed human RPE cells. Methods. Flow cytometry was performed on RPE cells that had been challenged wi th fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence. Re sults. Non-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. Th e kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures, FITC-ROS phagocy tosis proceeded at a fairly linear rate for the first 12 h in the 3 hu man cell cultures studied, but was rapid for the first 3 h before slow ing in the D407 cells. Within all cell populations, there was a hetero geneity of phagocytic activity which varied with time.Conclusions. Thi s automated technique for measuring phagocytosis is rapid, simple, hig hly accurate, avoids radiation hazards, and permits study of heterogen eity within cell populations. The biochemistry, physiology and pathoph ysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately q uantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor i ntensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large sta ndard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cy tometry, on the other hand, circumvents nearly all of these problems a nd may prove to be the optimal phagocytosis assay.