Did you change anything in-between the experiments e.g. ECM?

i didn’t culture the huvecs on any extracellcular matrix. Should I?

I used the method from Article A thin layer angiogenesis assay: a modified basement matrix ...

did you use matrigel (from Corning)? The thrid photo looks normal. Maybe you seeded too many cells. You can try seeding fewer cells and see what happens.

Ana Sayuri Yamagata yes I use Corning matrigel and sometimes geltrex

I did try seeding less cells but became like no1 and no2 photos.

How many times did you freeze and thawed the matrigel? More than 2 is not good.

we thawed once

Have you used growth factor-reduced Matrigel both times? Was the same final concentration of Matrigel in both experiments?

Its growth factor reduced matrigel. But different batches from Corning and geltrex (thermo fisher)