4.7. VEGF Binding Assays

RPE cells were seeded into 48 well plates at 20,000 cells per well. The cells were maintained overnight prior to placing the cells into the hypoxia chamber or maintaining the cells under normoxic conditions. The cells were subjected to hypoxia/normoxia for 72 h prior to conducting binding assays. For binding assays, the medium was removed and the cells were washed 3 times with ice cold binding buffer (25 mM Hepes pH 6.5, 1 mg/mL BSA, DMEM; 200 µL/well). A total of 100 µL of binding buffer was added to each well and the cells were incubated at 4 °C for 10 min. ¹²⁵I-VEGF (20 ng/mL) was added to each well and allowed to bind to the cells for 2 h at 4 °C, until equilibrium was reached. Unbound ¹²⁵I-VEGF was removed by washing the cells 3 times with ice cold binding buffer and the ¹²⁵I-VEGF that was bound to fibronectin and HSPG was released by exposing the cells to high salt buffer (25 mM Hepes, 2 M NaCl; 100 µL/well) for 5 s, followed by PBS (100 µL/well), and the radioactivity in the combined samples was counted in a Packard Instruments Auto-gamma counter.