Cultrex® In Vitro Angiogenesis Assay Tube Formation Kit

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	- Cells are coated with reduced growth factor basement membrane extract (RGF-BME).

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- Cells are coated with reduced growth factor basement membrane extract (RGF-BME).

Publication protocol

In vitro angiogenesis assay was performed to evaluate the effect of honokiol on the ability of hRMVECs to form tubes on basement membrane, under the trigger of pro-angiogenic factors (VEGF). To carry out this assay, in vitro angiogenesis tube formation kit was used (Trevigen, Gaithersburg, MD). A 96-well plate was quoted with 50 µL of reduced growth factor basement membrane extract (RGF-BME). RGF-BME was allowed to solidify by incubating at 37°C for 1 h. hRMVECs were plated slowly on the membrane at a density of 3×104 cells/well. The cells were treated with different treatment conditions. Cells in VEGF containing ENDO-growth media served as the positive control. The two negative controls used in this experiment were cells incubated in ENDO-basal media and ENDO-growth media with 100 µM sulforaphane. In order to evaluate the effect of honokiol on the tube formation, different concentrations of honokiol (2.5–40 µM) were added in the ENDO-growth media. Cells were incubated for 18 h at 37°C in a CO2 incubator. The media was carefully aspirated and cells were washed with 150 µL PBS. Further to each well 2 µM Calcein AM (in PBS) was added and incubated for 30 min at 37°C. After the incubation Calcein AM was removed and PBS was added. Extent of tube formation was assayed using fluorescence images. Efficacy of honokiol on tube formation was evaluated by quantitating the average branch length using angiogenesis analyzer plugin for ImageJ.


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