Fig. 3. Decreased phagocytosis of FITC-POS by CLN3 disease hiPSC-RPE cells.

A Schematic showing the protocol used to evaluate POS phagocytosis by hiPSC-RPE cells. Specifically, hiPSC-RPE cells were fed ~20–40 FITC-labeled POS/RPE cell for 2 h at 37 °C. Subsequently, any FITC-POS remaining on hiPSC-RPE cell surface was removed by washing with 1X PBS and the cells were fixed, immunostained with ZO-1 (red channel) and DAPI (blue channel) and the amount of POS phagocytosed by hiPSC-RPE cells was determined by measuring FITC fluorescence (green channel) using confocal microscopy. B–D Representative confocal microscopy images showing a similar pattern of ZO-1 (B) and DAPI localization (B–D), but decreased number of FITC-POS in CLN3 disease hiPSC-RPE cultures compared to control hiPSC-RPE cultures 2 h post-FITC-POS feeding (scale bar = 10 µm) (n = 4). Of note, panel D is the enlarged view of the highlighted area (red box) in panel C. E Quantitative analyses of FITC-fluorescence-labeled POS particles (particles < 5 µm, threshold set to exclude POS aggregates), 2 h post-POS-feeding, showing decreased number of phagocytosed (bound+ingested) FITC-POS per field of view in parallel cultures of CLN3 disease hiPSC-RPE cells compared to control hiPSC-RPE cells (n = 4, p = 0.003, two-tailed unpaired Student’s t-test). For the boxplot, + represents mean, center line represents median, box represents interquartile range between first and third quartiles, and whiskers represent 1.5* interquartile range. **p < 0.005.