Capillary tube formation assays were carried out as described by Qiu et al [52] (link). Briefly, HUVEC cells (3×104 cells/well) were cultured on Matrigel in a 96-well plate for 24 hrs with 100 µg/mL of P0-P5, P1C-P5C or blank control. The enclosed capillary networks of tubes were photographed by a microscope (Olympus, CKX41, Japan) and the numbers of capillary tubes formed were counted at different time intervals.
The transwell cell migration assay was performed by using a 24-well chamber (Costar, Cambridge, MA, U.S.A.) as the outer chamber and polycarbonate filters (8 µm pores) as the inner chamber. HUVECs were starved overnight in serum-free F12 medium, harvested by trypsinization and 5×104 cells were seeded into the inner chambers in F12 (1%FBS) media with 100 µg/mL P5 or blank control. The outer chambers contained the F12 media with 10% FBS. After incubation for 18 hrs at 37°C, the cells on the lower surface of the filter were fixed and stained with 0.1% crystal violet. Images of the migrated cells were taken using a microscope (Olympus, CKX41, Japan). The migrated cells were quantified at 595 nm after extraction with 10% acetic acid. Migrated cells in the outer chambers were also quantified independently by incubating the out chambers in F12 media with 10% FBS for 8 days followed by resazurin quantification.
The transwell cell migration assay was performed by using a 24-well chamber (Costar, Cambridge, MA, U.S.A.) as the outer chamber and polycarbonate filters (8 µm pores) as the inner chamber. HUVECs were starved overnight in serum-free F12 medium, harvested by trypsinization and 5×104 cells were seeded into the inner chambers in F12 (1%FBS) media with 100 µg/mL P5 or blank control. The outer chambers contained the F12 media with 10% FBS. After incubation for 18 hrs at 37°C, the cells on the lower surface of the filter were fixed and stained with 0.1% crystal violet. Images of the migrated cells were taken using a microscope (Olympus, CKX41, Japan). The migrated cells were quantified at 595 nm after extraction with 10% acetic acid. Migrated cells in the outer chambers were also quantified independently by incubating the out chambers in F12 media with 10% FBS for 8 days followed by resazurin quantification.
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