We have tried conducting tube formation assay for RF/6A (Choroid Retinal Endothelial Cells obtained from Rhesus species). Passage number is 46. We have used MEM (ATCC recommended) medium supplemented with 10% FBS and 1% Antibiotic Antimycotic solution.

We had used non-treated 96 well plates and coated 80 ul of Corning Matrigel Basement Membrane Matrix per well. We seeded the wells at different cell densities - 10k, 15k, 20k, 25k and 30k and observed it every hour about 12 h and then after 24h.

However, we did not observe tube formation in the well plate.

What could be the reason for this? How can we resolve this?