Hello! I am trying to make a monolayer using retinal endothelial cells. I followed the protocol I have for brain cells (which has worked for me in the past), but after adding FITC I found that the control group was the same as the damage inducer group, and figured I the monolayer didn't properly form. I plated the cells again in a transwell monolayer plate (hanging inserts), and I cannot seem to spot the cells under a microscope even after focusing. I have tried many different things, such as switching to standing inserts, increasing fibronectin cure time in incubator, switching from fibronectin to another coating, changing cell concentration, plating cells first and letting sit before adding the rest of the media, changing timepoint for reading from FITC (100uL from bottom well). however, NOTHING seems to be working. I am on a tight deadline and will deeply appreciate any help. Thank you!