2.6 Endothelial cell proliferation and migration assay

To assess the effect of medin on cellular proliferation, HUVECs were plated in a 96-well plate at 8000 cells per well. After 24 h, cells were treated with medin 5 μM or vehicle and proliferation assay was performed on cells treated on day 0 and 2 days following treatment using Alamar Blue Assay (Thermo Fisher Scientific) modified from established protocol.20 To perform the proliferation assay, media was removed from the well plate and the samples were washed three times with DPBS. The Alamar Blue solution was prepared in warmed culture media at concentration of 10% (v/v) and then added to each well followed by incubation for 4 h. Afterwards, fluorescence intensity was measured using a fluorescent plate reader (FLUOstar Omega, BMG LabTech, Ortenberg, Germany) at 544–590 nm wavelength.

A scratch-wound assay, modified from established protocol,21 was utilized to analyze the effect of medin on endothelial cell migration. HUVECs were plated in 24-well plate and incubated until 90% confluent. Later, a pipette tip (20 μl) was used to perform a straight scratch in the well. The wells were next washed with DPBS and replaced with cell culture media as a control or media containing medin 5 μM. The 24 well-plate was placed into a microscope incubator and imaged for 8 h overnight. Next, each time point was analyzed by making 10 distance measurements between the scratch. Distance migrated were quantified in ImageJ 1.49 analysis software.