I am evaluating the effect of certain compounds on endothelial cell tube formation using Matrigel reduced growth factor. I have performed other assays with these cells and their peak effect is after 24-48h. I see the cells already form tubes 4h after plating in Matrigel and the best imaging is 16-18h after plating. Has anyone exposed their cells to a compound of interest for a certain time, before plating in Matrigel? I am just worried that the exposure time is not long enough. My alternative is to use a higher concentration of ligands as the current levels are low. Unfortunately, I do not have access to non-Matrigel tube formation assays where you use a feeder cell (nice assays because the exposure time is a few days). Any advice would be appreciated.