Tryple select 10

The pHi of CEC SPs was measured by using the cell-permeable probe 3′-O-acetyl-2′,7′-bis(carboxyethyl)-5,6-carboxyfluoresceinacetoxymethylester (BCECF-AM special packaging; Dojindo Laboratories. Kumamoto, Japan). Cells were detached with TrypLE Select 10 × (Thermo Fisher Scientific) for 15 min at 37 °C, washed twice with the HEPES buffer (152-mM NaCl, 5-mM KCl, 5-mM glucose, 20-mM HEPES), and then incubated with BCECF-AM for 30 min at 37 °C in full humidity with 5% CO₂. Cells were washed twice with the HEPES buffer, and 150 µL/well of the cell suspension solution was poured into a 96-well plate. The fluorescence intensity was determined using a fluorescent plate reader with an excitation wavelength of 488 nm (GloMax Explorer; Promega Corporation, Madison, WI, USA). In this study, we used Dojindo’s assay kit (Dojindo Laboratories). The procedures for calibrating the BCECF fluorescence were described previously^{12 (link)}.

EVs were isolated in aseptic conditions in PBS using size exclusion chromatography as described in Section 2.2. The assay design was to ensure that the levels of exogenous EVs per treatment were close to the RPE physiological levels of EVs (modelling co‐culture conditions and 1:1 ratio of donor:recipient cells). The volumes of CCM, cell numbers that the CCM was collected from (donor RPE) and the amounts of exogenous EVs per treatment were therefore determined considering the cell counts of RPE subjected to the treatment with exogenous EVs (host RPE). Cells were grown on either 0.3 or 1.1 cm² Matrigel coated 0.4 μm PET hanging cell culture inserts (Merck) at a density of 450,000 cells/cm². Exogenous EVs or an equivalent amount of PBS for controls were supplemented to the exosome depleted RPE cell media in 12 treatments over the course of 32 days in three biological replicates. A quarter of a transwell insert for each treatment category was collected and fixed immediately in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for TEM ultrastructural analysis. The remaining cells were either fixed in 4% paraformaldehyde (Santa Cruz Biotechnology) for 60 min, washed in PBS and stored at 4°C until use or dissociated with TrypLE Select 10× (ThermoFisher) and collected for western blotting.

StemCO and diffCO were dissociated into single cells with TrypLE Select (10×) (Thermo Fisher Scientific) and cell aggregates were removed with a 20 μm cell strainer (pluriSelect Life Science). The cells were then suspended in SCM with 0.4% agar (CytoSelect 96-Well Cell Transformation Assay, Cell Biolabs) and 10,000 cells/well were seeded into 96-well culture plates according to the manufacturer. The wells were applied with 100 μL of SCM and incubated at 37 °C under 5% CO₂ for 7 days. The cells were counted by CyQuant GR Dye according to the manufacturer.