Biocoat angiogenesis system ec tube formation 96 well plates

Becton Dickinson (BD)-BioCoat Angiogenesis System-EC tube formation 96-well plates (BD Biosciences, Bedford, MA, USA) were used for migration assays. Cells from each group were plated at 2 × 10⁴ in 50 µl media in each well. The plates were incubated for 16–18 h at 37°C and then labeled with BD calcein AM fluorescent dye. The plates were imaged at 4× magnification using an Olympus BX53 microscope, DP72 digital camera, and CellSens imaging software (Olympus, Center Valley, PA, USA). The digital images were analyzed using WimTube image analysis software (Wimasis, Munich, Germany) to measure the following: (1) total tube length, which is the arithmetic sum of the length of whole tubular structures; (2) total tube, which measures the arithmetic sum of tubular structures between two branching points or a branching point and a loose end; (3) total branching point, which is the arithmetic sum of the point where three or more tubes converge; (4) total loop, which is the sum of all circular objects enclosed by the tubular structures; (5) mean loop area, which is the arithmetic mean of the area of all the loops; and (6) mean loop perimeter, which is the arithmetic mean of the border of the tubular structure of all loops. Three images were analyzed per oxygen and treatment group to calculate mean values.