Assay of VEGF secretion

ARPE-19 cells were grown to 100% confluence in 24-well plates, cultured in DMEM-F12 alone for 24 h, and then stimulated with 500 μL of NLR for an additional 24 h. For evaluation of the effects of inhibitors on NLR-induced VEGF release, serum-deprived ARPE-19 cells were incubated for 1 h in the absence or presence of inhibitor and then for 24 h in the additional presence of NLR. The culture supernatants were collected, centrifuged at 20,000 × g for 5 min at 4°C to remove debris, and frozen at –80°C until subsequent measurement of VEGF-A concentration with the use of a multiplex human cytokine assay system or an ELISA.