4.6. FITC-Dextran Permeability Assay

Permeability assays were performed using modifications to our previously described procedures [29]. Transwell, 24-well inserts (Corning) were coated with 7.5 μg/cm² FN1 for two hours at 37 °C, aspirated, and then dried for 30 min at room temperature. Top chambers were then seeded with 1 × 10⁵ HUVECs in 100 μL of VascuLife^TM VEGF endothelial media (Lifeline Cell Technologies) and incubated for 30 min at room temperature to avoid the accumulation of cells at the edge of the wells. The cells were then cultured for 24 h with an additional 200 µL and 1 mL of growth media in the top and bottom chambers, respectively. To improve the robustness of the assay, wells were seeded with an additional 1 × 10⁵ HUVECs and the media replaced. After another 24 h of culture the media was removed from top and bottom chambers and the cells gently washed two times with dPBS, once with endothelial basal media (Lifeline Cell Technologies), and serum starved for 2 h. The cells were then treated with 100 μM AXT107 or equivalent DMSO vehicle for 1.5 h followed by a 3 h incubation with 400 ng/mL Ang2, 10 ng/mL TNFα, and/or PBS control in the top chamber, and 25 μg/mL FITC-Dextran (40 kDa MW) in the bottom chamber. For detection, 10 μL samples were taken from the top chamber, diluted 1:10 in water, and the fluorescence quantified with a Biotek Synergy HT fluorescence plate reader (Biotek Instruments, Winooski, VT, USA).