Essen wound maker

Control and ELTD1 overexpressing HUVECS were seeded at 5 × 10⁵ cells per well into a 24-well ImageLock plate (Essen BioScience) and grown to confluence. Once confluent, a scratch defect was introduced into each well using a 4 pin Essen Wound Maker (Essen BioScience) and scratches were imaged every 30 min, using an Incucyte Imaging Incubator (5% CO₂ at 37 °C), until the defect had been repaired. The cell-free scratched images of each group at individual time points were then manually traced using the self-made stylus described above with the area quantified using FIJI.

For in vitro migration studies, cells were plated in triplicate on 96-well Essen Image Lock plates (Essen BioScience) at 40,000 cells per well. Cancer cells were pre-treated with 30 ng/ml Mitomycin C for 2 h to block cell proliferation. The Essen Wound Maker (Essen BioScience) was used to make the wounds in confluent cell culture monolayers. Cancer cells were then treated with either 25 or 50 μg ml⁻¹of collagen I (Invitrogen) after scratch. Wound closure was monitored by acquiring images every 1 h over a 24 h period with the Incucyte ZOOM. An integrated metric called relative wound density (RWD) was used to quantify effects on migration. The grey area indicates the “wound gap” (devoid of cells), while the orange area indicates the movement of cancer cells migrating toward the gap area.