Photoreceptor Outer Segment Phagocytosis

Purification and labeling of bovine photoreceptor outer segments (POS) was performed as described previously [26 (link)]. FITC-labeled POS were incubated with mcRPE at a ratio of 20 POS per cell and mouse IgG1 for 16 h at 37 °C and 5% CO₂. Microcarriers were subsequently washed five times with PBS+/+ and fixed using 4% paraformaldehyde. For function-blocking experiments, mcRPE were pre-incubated for 1 h with 30 ug/mL α_vβ₅ integrin antibody (Abcam, #ab177004) followed by a 16 h co-incubation of mcRPE with α_vβ₅ integrin antibody and FITC-labeled POS. Samples were rinsed, fixed and co-stained with phalloidin (Thermo Fisher Scientific #A22287) to label F-actin and Hoechst to label nuclei. Imaging was performed using a Leica SP8 Resonant Scanning Confocal microscope. POS particles and nuclei were thresholded and 3 representative field over-views were counted using Analyze Particles function with size restrictions; quantification was performed using FIJI ImageJ.