FIGURE 4.

Phagocytosis activity in ABC is higher than ARPE-19 cells. (A) Phagocytosis of photoreceptor outer segments (POS) or polystyrene microspheres (beads) by ARPE-19 and ABC cells over time. Confocal microscopy was used to analyze bound and internalized FITC-labeled POS or fluorescent beads (Supplementary Figure 8). The ratio of relative fluorescence intensities was determined by measuring total fluorescence intensity of the green signal from the added material (beads or POS) over the total blue signal (Hoechst). (B) Live images showing ABC cells fed with and without POS. (C) Confocal images (z-stacks) of ABC cells show uptake of (i,ii) human POS and (D) rat photoreceptor outer segments; orthogonal view displays co-localization of the FITC labeled POS (green) and opsin (red). (E) Immunoblots with opsin antibody against the lysates of ABC and ARPE19 cells fed with bovine POS (Supplementary Figure 9 shows two independent biological whole membrane western blots). (F,G) Assessment of POS phagocytosis in ABC cells by flow cytometry. After gating out cell debris and doublets (Supplementary Figure 4A), and not bound/internalized POS (F, bottom left) in control or cells exposed to FITC-POS, 7AAD versus FITC signals were obtained (F, Gated, 100,000 events per condition were assessed). The great majority of viable cells (7AAD negative cell population) are POS + (bottom right). (G) Analysis of FITC signal alone showed a shift in the peak of it in ABC cells fed with FITC-labeled POS compared to one of the control ABC cells (white). (H,I) Quantitative analysis of Hoechst-stained ABC cells by fluorescence microscopy of POS phagocytosis effects on apoptotic cell population after oxidative stress. (H) Analysis of apoptosis by Hoechst staining. (I) Quatification of panel (F). (A,H,I) One Way ANOVA and Tukey’s HSD for pairwise comparisons was applied. Results represent averages ± SEM of repeats of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.