Tube-formation assay
250 µl Matrigel (Corning) was added to each well of a 24-well plate for
overnight. Then, 7-day-cultured ECs were plated with a density of
5×104/well above Matrigel and cultured in 500 µl EGM-2
medium in an incubator[23-25]. After 48 hours, tube formed by ECs
were observed with a light microscope (Olympus) and 3 photographs per
well were taken randomly[23-25]. ImageJ
(National
Institutes of Health, MD, USA) was used to quantify the relative tube
length.