4.6. Trans-Endothelial Electrical Resistance (TEER)

The effect of recombinant flavivirus NS1 proteins on endothelial permeability was evaluated by measuring TEER of EC monolayers grown on a 24-well Transwell polycarbonate membrane system (Transwell^® permeable support, 0.4 μm, 6.5 mm insert; Corning Inc., Corning, NY, USA), as previously described [8,19]. Briefly, HUVECs or HBMECs (80–90% confluency) cultured in vented 75 cm² flasks (Corning^®) were detached using a combination of three washing steps using sterile 1× PBS supplemented with EDTA (2 mM) (lifting buffer), followed by two additional washing steps (~30 s total) using a solution of trypsin-EDTA (0.25%) (GIBCO, Thermo Scientific, Waltham, MA, USA). Detached cells were resuspended using fresh culture medium and then counted using a tissue-culture hemocytometer. A total of 1 × 10⁵ cells in 300 uL of medium were seeded on the apical side of Transwell inserts (top chamber) placed in a 24-well plate containing 1.5 mL of EC culture medium (basolateral side). Transwells containing ECs were incubated at 37 °C and 5% CO₂ for 3 days, and 50% of culture medium was changed in each well every day post-seeding. Cells were grown until TEER values between 150 and 180 Ohms (Ω) were reached, depending on cell type, indicating 100% cell confluency. Individual flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein) were then added to the apical side of the Transwell insert (top chamber, 300 μL) containing the cell monolayer. TEER values, expressed in Ohms (Ω), were collected at sequential 2-h time-points over 3–11 h following the addition of test proteins using an Epithelial Volt Ohm Meter (EVOM) with a “chopstick” electrode (World Precision Instruments). Inserts with no cells containing medium alone were used as negative controls to calculate the baseline electrical resistance. Endothelial permeability was expressed as relative TEER, which represents a ratio of resistance values (Ω) as follows: (Ω experimental condition—Ω medium alone)/(Ω non-treated endothelial cells—Ω medium alone).