Serotonin and 5-Hydroxytryptophol Regulate HUVEC Migration

The cell migration assay was performed following the protocol previously described in Cerezo et al. [19 (link)]. The HUVECs were placed onto 50 mm imaging dishes (Ibidi, Martinsried, Germany). Once cells reached full confluence, the monolayer was scratched across the centre using a sterile 200 μL pipette tip. Subsequently, the cells were then rinsed twice with PBS to eliminate any dead cells. Serum-free medium containing 1 mM serotonin, 1 mM 5-hydroxytryptophol or vehicle controls were added (final concentration ≤ 0.1% DMSO) for 4 h prior to VEGF stimulation (25 ng/mL) over 24 h. A phase contrast microscope (Nikon, Tokyo, Japan) was used to photograph the wound-gaps. Nis-Elements BR v.4.30.02 software (Nikon, Tokyo, Japan) was utilised to measure the initial and final sizes of each wound. The following formula was used to quantify the migration distance: initial wound size minus final wound size divided by two. Two independent experiments were performed.