I am trying to optimise my migration assay for melanoma cell line. I am seeding 100k cells per 8um insert and leaving 6-24 hours for migration, then fixing in methanol for 20 mins, followed by 1% CV stain for 15 mins. I clean the inserts gently with a cotton bud to remove cells inside insert and wash in water to thoroughly remove CV. The problem I have is that cells are migrating at the edges of the insert or in patches, causing large range in cell counts that affect significance of my data. I have tried gentle agitation, tilting the inserts back and forth, pipetting cells thoroughly to ensure they are not clumping, but still the same problem persists.. Attached is 2 images taken from the same membrane, from the edge and from the middle, you can see how different they are.