Aim for 95-100% confluent monolayers around the cell exclusion zone at the start of the experiment. Seeding at a higher density may result in cells in the Detection Zone at the beginning of the assay while seeding at a lower density will yield fewer migrating cells in the detection zone after migration. Typically, 20,000 – 75,000 cells are seeded per well in Oris and Oris Pro 96-well plates, and 2,500 – 10,000 per well in Oris Pro 384-well plates. For your first experiment, we recommend testing several different cell seeding densities as described in Appendix 1 in the assay protocol. For Oris 3D Embedded Invasion assays, we recommend seeding at 30,000 – 50,000 cells per well. As many as 75,000 cells per well may be seeded for some cell lines, but many invasive cells lines express high levels of matrix metalloproteinases that can degrade the collagen to the point where it collapses or even liquefies at high cell densities. Adding fewer cells ameliorates this problem.