Permeability assay

The permeability of the barrier to Sodium Fluorescein (NaF) was studied. Fifty microlitres of a solution containing 0.5 mg/ml NaF in PBS was added to the luminal side of each TW insert 1 min prior to PEFs application. Immediately after PEFs application, the plates were placed in a darkened incubator for 20 min with mild agitation. The fluorescence of the medium collected from the basal compartment was measured using TECAN pro200 plate reader (485/538 nm excitation/emission). In order to study the recovery of the barrier, the permeability assay was repeated 24 h later in the same inserts (the medium was replaced with fresh medium after the 20 min permeability assay). N = 6–12 TW inserts for each voltage amplitude.

The endothelial permeability coefficient (Pe) in cm/min was calculated as described in Vandenhaute et al. (Vandenhaute et al., 2012). In short, the clearance principle was used to obtain a concentration-independent transport parameter. The average volume cleared was plotted versus time, and the slope was estimated by linear regression. Both insert permeability (PS_f, for insert only coated with Matrigel) and insert plus endothelial cell permeability (PS_t, for insert with Matrigel and cells) were taken into consideration according to the following formula:

To obtain the endothelial permeability coefficient (Pe) of the molecules (in cm/min), the permeability value for the endothelial monolayer (Ps_e) was divided by the surface area of the porous membrane of the insert.