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An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis

m, 574625) was used to generate dose response curves from 0.3 to 160 μM. Suramin was solubilized in H2O and serially diluted into basal media containing 0.1% BSA.

After 16-18h, the media was discarded and cells were washed twice with Hanks Balanced Salt Solution (HBSS) and stained with Calcein AM (Invitrogen, C-3100MP), 8 μg/mL for 30 minutes at 37C/ 5% CO 2. The dye was discarded and cells were washed twice to remove excess dye. Some preparations were fixed with warmed 1% paraformaldehyde (in HBSS), washed twice with PBS and labeled with Alexa 488 Phalloidin (Invitrogen, A12379, 1:200 dilution).

Cells were imaged on the BD Pathway Bioimager in confocal and non-confocal mode using the 4 (NA 0.13) objective for quantification of tubule formation and 10 (NA 0.3) to generate higher resolution images.

Results

Initial results suggested that the tube formation event occurs across several focal planes, even with the low magnification used. This is not surprising considering that the endothelial cells are known to actively migrate into the BD Matrigel Matrix. We therefore employed a new feature of the BD Pathway Bioimager to automatically generate a single image representing the in-focus portion of multiple Z sections across a plate. This collapsed stack feature was combined with the ability of the system to acquire confocal images, thus ensuring a high degree of optical sample flatness even before the image was analyzed. Image analysis was conducting using the new BD Tube Formation Module, an advanced development of the BD Neurite Outgrowth Module (Cat. No. 346203). The algorithm thresholds the cells from the background and identifies a number of features including, the total length of the tubules, the total area of the tubules and the total number of segments. The software allows both turn-key analysis using default values as well as adjustment of parameters for t
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