*4.9. VEGF ELISA*

ARPE-19 and RPE supernatants were collected after treatment with LH extracts (1, 10, 50, and 100 μg/mL) on Day 3 after stimulation [11]. Medium with sulfated fucan was exchanged 24 h (ARPE-19) or 4 h (RPE) before collecting supernatants. Human VEGF DuoSet ® ELISA was used for ARPE-19, whereas the Human VEGF Quantikine ® ELISA was used for RPE supernatants (both R&D Systems, Wiesbaden, Germany). ELISAs were performed according to the manufacturer's instructions. Untreated cells and medium samples were tested as controls. To set the VEGF secretion in relation to the cell viability, MTT assay was conducted after collection of the supernatants on Day 3.
