Figure 3.

Laser scanning confocal microscopy and quantitative fluorescence imaging of FITC-ROS phagocytosis by RPE-J cells. (A, B, D, E, G, H) Confluent RPE-J monolayers were challenged with FITC-ROS for 2 or 5 h, fixed, and nuclei were stained with propidium iodide. (B, E, H) fluorescence of extracellular ROS was quenched with trypan blue. (C, F, I) RPE-J cells were challenged with unlabeled ROS for 2 or 5 h, fixed, and rhodopsin stained with Cy3 secondary antibody before permeabilization and with FITC secondary antibody after permeabilization. ROS accessible for labeling before permeabilization appear in yellow due to double staining, while internalized ROS appear in green. Note that after 5 h of phagocytosis, some internalized ROS have been lysed as indicated by a dispersed intracellular fluorescence. (J–L) Fluorescence imaging quantification. The same filter used to obtain the confocal images were used for quantification (K). Representative fields of constant areas (rectangles in J) were scanned; fluorescence intensities are shown as bar graph in L. Fluorescence derived from FITC-ROS and immunolabeled ROS were within 10% of each other. (Bars = 5 μm.)