4.7. VEGF Analysis

Cells were treated as indicated in the figure legends. Medium was collected following 10 h treatment. VEGF was analyzed using a VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA). An experiment for each condition was carried out in triplicate. To measure tissue VEGF level, the tissue homogenates prepared using buffer C were centrifuged at 400 g and 4 °C for 2 min. The supernatants (100 μL) were transferred to fresh microtubes and then centrifuged at 16,800 g and 4 °C for 10 min. An appropriate volume of the supernatant was subjected to ELISA. The protein amount in each sample was determined to normalize VEGF levels.