In Vitro Angiogenesis Assay Kit

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	- Cells are seeded with Endothelial Cell Basal Media (EBM-2) supplemented with Endothelial Cell Growth Supplements (EGMTM-2)

Protocol tips
- Cells are seeded with Endothelial Cell Basal Media (EBM-2) supplemented with Endothelial Cell Growth Supplements (EGMTM-2)

Publication protocol

In vitro angiogenesis assay was performed and evaluated according to the instructions of in vitro Angiogenesis Assay Kit (ECM625; Millipore, Billerica, CA, USA). U87-shGMF-β or U87-mock cells (5 × 103 per well, 96 well size) were seeded on the polymerized Matrigel, and incubated in Endothelial Cell Basal Media (EBM-2) supplemented with Endothelial Cell Growth Supplements (EGMTM-2; LONZA, Walkersville, MD, USA). Then, tube formation in each group was inspected under a phase contrast fluorescent microscope (BX51 CKX41-F32FL, Olympus, Tokyo, Japan). The formation of tubules is a dynamic process, starting with cell migration and alignment, followed by the development of capillary tubes. The quantification of tubulogenesis followed two evaluation criterions, “pattern recognition” and “branch point counting”. According to “pattern recognition” evaluation, a numerical value was assigned to each pattern: 0, individual cells; 1, a queue of cells; 2, capillary tubes; 3, sprouting capillaries; 4, closed polygons; 5, complex mesh like structures. Thus, a numerical summation was scored in each view-field. By the criterion of “branch point counting”, the newly formed branch was defined as an isolated cellular line sprouting from existing capillary tubes. Branch points were counted and totaled in each view-field. Five random view-fields per well were observed at 100 × magnification, then the quantification values were averaged in each group.


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