Mmp inhibitor 2

For all treatments, 75k cells endothelial cells were plated to gelatin-coated 12 well plates. 24 h later the endothelial cells were washed twice with 1×PBS, and placed in a low-serum media composed of basal EBM-2 endothelial cell media (Lonza) containing 0.5% FBS for 16 h before treatment as indicated in figure legends. For treatment with cell culture supernatants, ovarian cancer cells were grown to sub-confluence, and placed in normoxia or hypoxia (1.5% O₂) for 24 h. Cell culture supernatants were cleared by centrifugation and mixed with low-serum EBM-2 at a 1:1 ratio and placed on endothelial cells for 24 h. For VEGF-A neutralization, 5 μg mL⁻¹ anti-VEGF-A antibody (R&D) or isotype was added to cell culture supernatants for 30min at room temperature prior to addition to HMVEC cells. In cases of Aspirin treatment, 200 μM Aspirin was added to cells during exposure to normoxia or hypoxia. For treatments with ascites, samples were mixed with low-serum EBM-2 at a 1:1 ratio and placed on endothelial cells for 24 h. To enhance surface expression of FasL for flow cytometry, a broad spectrum metalloproteinase inhibitor (MMP Inhibitor II, 50nM, EMD Millipore) was added concurrently. For flow cytometry, HMVECs were washed twice with 1× PBS following treatment and detached with 1×PBS containing 2 mM EDTA at 37C for 5-10 min.