4 kda fitc dextran solution

To assess BBB permeability, 100 μg/mL 4 kDa FITC–dextran solution (Merck-Sigma-Aldrich, Overijse, Belgium) was added to the upper compartment of the insert in the static BBB model. For the dynamic BBB model, the membrane was first removed from the TripleB slide and transferred to the custom 6-well transwell which was placed in a 6-well plate. Subsequently, the same concentration of FITC–dextran solution was added to the upper compartment of the custom built transwell (100 μg/mL). The fluorescence recovery in the lower compartment was measured after 60, 120, and 180 min using a Victor3 multilabel fluorometer in both models. As a positive control, 100 μg/mL FITC–dextran was directly added into the lower chamber of the static and the dynamic BBB model and was compared with a cell-free insert. The negative control consisted of medium only. The apparent permeability coefficient, Papp, was evaluated according to the following equation: $$\mathrm{Papp} \left(\frac{\mathrm{cm}}{\mathrm{s}}\right)=\frac{\mathrm{dQ}}{\mathrm{dt}} \times \frac{1}{\mathrm{A} \times {\mathrm{C}}_0}$$
where dQ/dt is the amount of FITC–dextran present in the basal compartment as a function of time (nmol/s), A is the surface area of the membrane (0.33 cm² for static BBB and 0.32 cm² for TripleB) and C₀ is the original concentration of FITC–dextran added in the upper chamber at the start of the experiment (nmol/s) [45 (link),46 (link),47 (link),48 (link)].