Sodium-fluorescein permeability measurements

The paracellular barrier function of the endothelial cells was evaluated by measuring apparent permeability of cells to sodium-fluorescein (Na-Fl), which is a hydrophilic fluorescent molecule (MW: 376 Da). Firstly, the cells were washed with Ringer HEPES buffer, then Na-Fl was diluted to 10 µg mL⁻¹ with Ringer HEPES buffer. It was placed onto the luminal side of the insert and left at 37 °C for 1 h. After 1 h, samples were removed from the abluminal side and replaced with Ringer HEPES buffer. The Na-Fl concentration was obtained using a fluorescence spectrophotometer (FluoroskanAscent™, Thermo Fisher Scientific, France), with excitation (485-nm) and emission (530-nm) wavelengths. Papp is measured in cm s⁻¹ and is calculated using the following formula:

where P_app is the apparent permeability, V_r is the volume of medium in the abluminal side, C₀ and C₁ are the concentration of fluorescent compound in the luminal chamber at t₀ and in the abluminal side after t time, i.e., 1 h of incubation, and S is the monolayer’s area⁵⁶.