8.2. Invitro network angiogenesis assay

To study the effect of various concentrations of MGCD0103 on the inhibition of angiogenesis, we achieved network angiogenesis in vitro by using the Co-Culture Tube Formation Assay kit (Ibidi, Gräfelfing, Germany) as described in the company’s instructions. The Matrigel (10 μL) was placed in a 15-well chamber slide and incubated with 5% CO₂ at 37 C to solidify for 1h. Each of the C6 and T98G cells at a density of 2.5 ×10⁴ was separately mixed with 10⁴ human mammary epithelial (HME) cells as co-culture, then they were added to each tube containing 50 μL RPMI and incubated for 6 h to network formation. After the culture medium was aspirated off the Matrigel surface, 50 μL of growth medium supplement of different concentrations was added to each well-containing network. The chamber slide was incubated for 24 h in 5% CO₂ and 37C. After that, the culture medium was expelled. Cells were fixed with methanol and the image was observed by light microscopy (Leica Microsystems, Inc, USA). The tube information was counted by Image J software (National Institutes of Health, Bethesda, MD, USA).