Spectramax id3

EV-derived Aβ HiLyte fluorescence was quantified in the recipient NPCs using a plate reader. Briefly, NPCs were plated in 96-well black plates (9,000 cells/well) and differentiated for 3 days with the last 24 h in the presence of the isolated EVs. To block Serpine-1 activity, selected NPC cultures were cotreated with 2 μM PAI039 and brain endothelial EVs for 24 h. After treatment, cells were washed with PBS, fixed with ethanol for 30 min at 4°C, washed again with PBS and Aβ HiLyte fluorescence was measured with a plate reader (Molecular Devices, SpectraMax iD3) as suggested by the manufacturer (Anaspec, Ex/Em 503/528 nm). Cell nuclei were stained with DRAQ5 (Cell Signaling, Catalog #4084L, dilution 1:1000) for 5 min, washed with PBS and DRAQ5 fluorescence was measured with the same plate reader (Ex/Em 647/681). Aβ HiLyte fluorescence was then normalized to nuclear DRAQ5 fluorescence.

EV-derived Aβ HiLyte fluorescence was quantified in the recipient NPCs using a plate reader. Briefly, NPCs were plated in 96-well black plates (9,000 cells/well) and differentiated for 3 days with the last 24 h in the presence of the isolated EVs. To block Serpine-1 activity, selected NPC cultures were cotreated with 2 μM PAI039 and brain endothelial EVs for 24 h. After treatment, cells were washed with PBS, fixed with ethanol for 30 min at 4 °C, washed again with PBS and Aβ HiLyte fluorescence was measured with a plate reader (Molecular Devices, SpectraMax iD3) as suggested by the manufacturer (Anaspec, Ex/Em 503/528 nm). Cell nuclei were stained with DRAQ5 (Cell Signaling, Catalog #4084L, dilution 1:1000) for 5 min, washed with PBS and DRAQ5 fluorescence was measured with the same plate reader (Ex/Em 647/681). Aβ HiLyte fluorescence was then normalized to nuclear DRAQ5 fluorescence.