Endothelial Tube Formation Assay

Endothelial tube-like formation was assessed, as previously described^{21 (link)}, using an angiogenesis kit (Abcam) and following manufacturer’s instructions. Briefly, bovine lung microvascular endothelial cells (BLMVEC) (kind gift from Dr. Theresa Curtis, State University of New York at Cortland) were plated on wells coated with extracellular matrix, provided in the kit, in a 96-well plate and incubated with DMEM + 1% FBS (negative control), MiDC CM, or DMEM + 1% FBS with 40 µM suramin, an inhibitor of angiogenesis, for 8 h at 37 °C. BLMVEC were washed, stained with an FITC fluorescent dye, provided in the kit, and imaged using a ZOE fluorescent imager (BioRad) and phase-ring microscope (Olympus, Tokyo, Japan). Three phase-ring images from each well were analyzed using the Angiogenesis Analyzer plug-in^{70 (link)} for ImageJ. Each test condition was run in duplicate wells.