Fluorescein isothiocyanate labeleddextran fitc dextran

Conventional in vitro BBB modeling was conducted by culturing primary HBMVECs grown in 2D as a flat monolayer by following the published protocols [35 (link)]. Permeability across the monolayer of HBMVECs was measured by using transwell units (24-well format, 0.4 µm pore size with high pore density PET track-etched membrane, cat # 353495), which were made to fit in a 12-well plate, purchased from Corning Incorporated, Corning, NY, USA. To create the BBB model, 1 × 10⁵ cells were seeded onto the transwell inserts and cells were allowed to grow for 2 days in complete classic medium. After endothelial cells become confluent on the transwell, cells were treated in fresh culture medium with 10 µg/mL of either Patient-Exo or Control-Exo for 24 h. The cell monolayer’s ability to limit the penetration of a high molecular weight compound was measured by adding 10 µg/mL fluorescein isothiocyanate-labeled dextran (FITC-dextran; molecular weight: 150 kDa, Sigma-Aldrich, St. Louis, MO, USA) to the culture transwell inserts. In the absence of primary HBMVEC, FITC-dextran added to the culture inserts served as controls. After incubation for 10, 30, 60, and 120 min, 20 µL of medium was collected from the lower compartment, and the fluorescence was measured with a spectrophotometer (BioTek Instruments, Winooski, VT, USA) set to 485/20 nm excitation and 528/20 nm emission.