Transwell polycarbonate membrane system

Human umbilical vein endothelial cells monolayers were grown on a Transwell polycarbonate membrane system (Corning Inc.) and treated with recombinant human IL-1β or preincubated with IL-1RA for 24 h. Half of the medium contained in the upper and lower chambers was replaced by fresh endothelial cell medium. Untreated HUVECs were used as a negative control, and medium alone was used as a blank control. Endothelial permeability was evaluated by measuring trans-endothelial electrical resistance (TEER) at 2-h intervals using the EVOM2 epithelial Voltohmmeter (World Precision Instruments). Relative TEER was expressed as follows: [(resistance in experimental group) – (resistance in medium alone)]/[(resistance in untreated HUVECs) – (resistance in medium alone)]; resistance was expressed in ohms.

As described previously [25 , 38 (link)], to evaluate the effects of MMP9 on the integrity of the SCB, 2.5 × 10⁵ primary mSCs were plated on a 12-well Transwell polycarbonate membrane system (Corning Life Sciences, Corning, NY, USA) with a 0.4-μm pore size and a 1.12-cm² membrane surface. The integrity of this in vitro SCB model was determined by measuring TEER using an epithelial volt/ohm meter (Evomx) with “chopstick” electrodes (World Precision Instrument) every day from day 2 after seeding. Experiments were performed with a high resistance until TEER values crossed 50 Ω • cm², depending on the cell type, indicating 100% cell confluency [38 (link)]. Subsequently, cells were infected with ZIKV (MOI = 5) and then treated with or without the specific MMP9 inhibitor JNJ0966 for 1h. The inserts were gently washed three times with medium to remove unbound virus, and cells were treated again with the inhibitor. The TEER was measured 12, 24, 36, 48, and 72h after infection. In addition, the activated MMP9 protein with or without JNJ0966 was added to the upper chambers, and resistance values were measured at the indicated times. The SCB permeability was expressed as the relative TEER, which represents the ratio of resistance values (Ω) as follows: (Ω experimental condition –Ω medium alone) / (Ω untreated SCs –Ω medium alone).