Scratch Wound Migration Assay of Human Umbilical Vein Endothelial Cells (HUVECs) for Investigating Angiogenesis In Vitro

To begin, seed 100 microliters of HUVEC cells into the wells of a 96 well plate. Place the plate in a cell culture incubator at 37 degrees Celsius under 5%carbon dioxide for six hours to facilitate cell settling. Next, replace the medium in each well with 100 microliters of endothelial cell growth basal medium, containing 2%FBS, and incubate again.

The following day, position the plate in a 96 well wound maker tool. Press down the lever of the device to generate a scratch. Carefully lift off the lid of the wound maker tool before releasing the lever to prevent double scratching.

Wash the wells two times with 200 microliters of FBS-supplemented endothelial cell growth basal medium. After confirming total debris removal, pipette 100 microliters of the prepared stimulation or control solutions into each well. Use a live cell imaging microscope to acquire images every hour for 24 hours.

The positive control showed successful migration in the original scratched area. Technical issues were seen as patchy cell growth, double scratching, or waves in the scratch borders. A 25%increase in relative wound distance was considered optimal.

Incorrect separation of the positive and negative controls indicated low dynamic range.