Cell permeability assay using FITC-dextran

Human SCEC were prepared and treated using the same method for TEER measurement as described above. Transwell permeability assays were carried out as previously described⁵⁴. In brief, 4 kDa, 70 kDa and 150 kDa fluorescein isothiocyanate (FITC)-conjugated dextran (FD) (Sigma) was applied at 1 mg/ml to the basal compartment of the transwells. Sampling aliquots of 0.1 ml were collected every 15 min for a total of 120 min from the apical side for fluorescence measurements and the same volume of culturing media was added to replace the medium removed. FITC fluorescence was determined using a spectrofluorometer (Optima Scientific) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Relative fluorescence units (RFU) were converted to values of nanograms per millilitre using FITC-dextran standard curves, and were corrected for background fluorescence and serial dilutions over the course of the experiment. The apparent permeability co-efficient (P_app, cm/s) for each treatment was calculated using the following equation:

where dM/dt (μg/s) is the rate of appearance of FD on the apical side from 0 min to 120 min after application of FD. C₀ (μg/ml) is the initial FD concentration on the basal side, and A (cm²) is the effective surface area of the insert. dM/dt is the slope calculated by plotting the cumulative amount of (M) versus time.