I use 50 µL of Matrigel (stored at 4°C overnight before use) in a 96-well plate, and all reagents are stored either at -20°C or 4°C. The Matrigel is incubated at 37°C for 30 minutes to 1 hour before seeding 20,000 cells, which are then monitored at different time points (up to a maximum of 10 hours).

Here are some images from my different trials.

I am desperately trying to perform a tube formation assay with primary HUVECs (Human Umbilical Vein Endothelial Cells) in the presence of anti- and pro-angiogenic factors. However, I am unable to obtain a clear network of tubes.

If anyone has experience with this type of assay or with HUVECs, I would greatly appreciate any suggestions to help improve my results. Thank you!