Quantifying Phagolysosome Dynamics in RPE Cells

Phagolysosomes were stained and imaged as previously described^{19 (link)}. Freshly dissected posterior eyecups were incubated at 37 °C for 15 minutes in DMEM (11995-065, Gibco) with 0.4 µM LysoTracker^TM Red DND-99 (L7528, Molecular Probes) to label acidified phagolysosomes and 5 µM 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) to stain nuclei. Confocal images (z-stacks, 0.3 µm steps, 30 µm in total) were captured in the central part of the RPE, using an Olympus IX-81 inverted microscope equipped with a Yokagawa CSU X1 spinning disk confocal scan-head, a Hamamatsu EMCCD camera (C9100-13). The volume occupied by phagolysosomes inside individual RPE cells was calculated using Volocity™ software (version 6.3, PerkinElmer). RPE cells were imaged from 8 independent experiments (4 WT and 4 TG animals) and a total of 13 microscope fields per group (3–4 images per animal), representing 297 and 331 RPE cells from WT and TG animals respectively, for final analysis. Mann-Whitney U-test (one-tailed) was used; significance was set to P ≤ 0.05.