In Vitro Sodium Fluorescein Permeability Assay

Briefly, bEnd.3 cells were seeded onto polycarbonate 12-well transwell inserts with a 0.4-μm mean pore size and a 0.33-cm² surface area (Corning, USA) at a density of 4 × 10⁴ cells/cm², and the growth medium was refreshed every other day. The seeded cells were allowed to grow at 37 °C in a 5% CO₂/95% air atmosphere until they reached 70–80% confluence, which was confirmed using phase contrast microscopy. Then, cells were cultured with different ACMs for 24 h. After two washes with PBS, the media in the transwell inserts were replaced with media supplemented with 500 μL of 1 mg/mL sodium fluorescein (NaF), the lower chamber was filled with 1000 μL of normal media, and cells cultured for 1 h at 37 °C under normoxic conditions. Relative fluorescence passing through the chamber was measured as follows: 100 μL of medium in the lower chamber was assayed in triplicate in black 96-well plates (Corning, USA). The fluorescence intensity was measured using an EnSpire Manager (PerkinElmer, USA) multimode plate reader at an excitation wavelength of 460 nm and an emission wavelength of 515 nm [11 (link)]. NaF permeability (μg/cm²) = total NaF quantity in the lower chamber/the surface area of insert (0.33 cm²).