Melanin content assay

To determine the melanin content, melanocytes were treated with 10 ng/ml VEGF165 for 48 h with or without incubation of 1 mg/ml TA, 5 μg/ml VEGFR-1 and/or VEGFR-2 neutralizing antibody, after which they were washed with PBS and harvested. After centrifugation at 18,000 × g for 5 min, the harvested cell pellets were solubilized in boiled 1 N NaOH (65 °C) for 2 h, and color was analyzed at 405 nm using a microplate reader. Melanin content was expressed as a fold change of the treated group vs. the control group.