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


Formation of new blood vessels is fundamental to the development of cancerous cell masses and has therefore been the focus of many drug screening and cell signaling research studies. Blood vessel development is a significant event in the development and growth of solid tumors, and is implicated in wound healing, retinopathy and macular degeneration.

Traditional animal models to quantify the degree of blood vessel formation are being replaced by cell culture assays that are easier to set up, statistically reliable and can be automated in a drug screening laboratory.1,2

These assays rely on the endothelial cells ability to form distinct blood-vessel-like tubules in an extracellular matrix (BD Matrigel Matrix) where they can subsequently be visualized by fluorescence microscopy. Although quantification of the tubules can be performed by manual tracing, this method precludes the use of the assay in unbiased high-throughput applications. Much preferred is the use of an automated confocal imaging system such as the BD Pathway Bioimager with sophisticated image and data analysis algorithms. The assay is designed to work with live cells in order to avoid fixation artifacts such as the disruption of tubules.


Human Umbilical Vein Endothelial Cells (BD HUVEC-2, Cat. No. 354151) were maintained in EGM-2 (Cambrex, CC-3156). The cells were washed and trypsinized (trypsin- EDTA, Cambrex, CC-5012) then resuspended into basal media (Cambrex, CC-3162) containing 0.1% BSA (Cat. No. 354331) and seeded into BD BioCoat Angiogenesis System: Endothelial Cell Tube Formation plates (Cat. No. 354150) at 20,000 cells/well and incubated for 16 to 18h at 37C (5% CO 2 ). These plates contain an optimized BD Matrigel Matrix which promotes endothelial cell tube formation. Suramin (Calbiochem, 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.


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 the development of specific assays. The software allows cellular debris or non-connected tube fragments to be removed prior to data analysis. The data generated is contained in text files and easily analyzed using BD Image Data Explorer (Cat. No. 341039).

Control images ( Figure 3 , left panel) obtained with the BD Pathway Bioimager show distinct development of tubes indicating that endothelial cells indeed do resemble blood vessels. The image in Figure 1 was acquired using a 10 objective and shows very clear development of endothelial cell-derived tubules. These tubules mostly originate at aggregates of cells and appear to actively connect with other tubules or aggregates. The total number of tubes formed ( Figure 3 and Figure 4 ) is an important measurement parameter since the underlying signaling and gene expression processes which mediates tube formation may offer an attractive leverage point for drug development.


The advent of automated imaging platforms such as the BD Pathway Bioimager allow the migration of microscopebased assays into the drug discovery and systems biology arena. For this to succeed, images not only need to be acquired in a highly reproducible and hands-off manner but must also be analyzed with minimal user intervention. We present here such an assay consisting of human endothelial cells, 96-well coated plates, an automated imaging system and data analysis software. Confocal microscopy combined with the collapsed stack feature of the BD Pathway Bioimager enables analysis of angiogenic tubules in a 3 dimensional matrix that better resembles the native physiological environment. Alternative methods using non-confocal systems are being used where the matrix is dried out overnight to collapse the cells into a flat plane this can not be done on live cells and may create significant distortions in the fragile branching structure of the sample.

Blood vessel formation is an important target in the treatment of multiple diseases including several cancer types. In the small molecule screening phase of drug discovery, animal experiments are low throughput and often technically too challenging, expensive and ambiguous to be used. Scoring the degree of blood vessel formation using an unbiased microscope approach allows this assay to be used in secondary screening applications against hundreds or thousands of putative drugs. Although cell culture experiments can currently not fully replace animal experiments, they aide in reducing the number of compounds to be tested and thus accelerate drug development and our understanding of cellular signaling events.


1. Auerbach, R., Lewis, R., Shinners, B., Kubai, L., Akhtar, N. 2003. Angiogenesis Assay: A Critcial Overview. Clinical Chemistry. 49:1, 32-40.

2. Taraboletti, G., Giavazzi, R. 2004. Modelling approaches for angiogenesis. EJC . 40, 881-889.

3. Fu, X.W, Nurse, C.A., Cutz, E. 2004. Expression of functional purinergic receptors in pulmonary neuroepithelial bodies and their role in hypoxia chemotransmission. Biochem. J. 385 (3-4), 275-284.



Page: All 1 2 3 4

Related biology technology :

1. Caspase-3 Activation - An Indicator of Apoptosis in Image-Based Assays
2. A Positive Selection Assay for Mutation Analysis in Big Blue Animals
3. Luminol-enhanced Assay for Superoxide Anion (O2 face="Symbol" )
4. Organize Your Data from the Big Blue Mutation Assay
5. Fluorescence-Based Single-Tube Assays to Rapidly Detect Human Gene Mutations
6. RNase Protection Assay (RPA) Using DIG-Labeled RNA Probes
7. measuring cell proliferation with SpectraMax M5, M2 and Gemini microplate readers using the CyQUANT Cell Proliferation Assay
8. Matched siRNAs and Assays: Ambion + Applied Biosystems = RNAi Success
9. Using Validated siRNAs in Functional Genomic Assays
10. Nuclease Protection Assays: The Basics
11. RNArticles: RNA Amplification Used to Develop a New In Vitro HCMV Assay
Post Your Comments:
(Date:10/9/2015)... ... October 09, 2015 , ... From blood to ... separating those cells from their surroundings for research, diagnostics, and cell therapy—also known ... address this, Ann Arbor-based startup Akadeum Life Sciences is developing a ...
(Date:10/8/2015)... Oct. 8, 2015   Intrexon Corporation (NYSE: ... announced the appointment of Joseph L. Vaillancourt ... Nir Nimrodi who continues in his role ... Intrexon,s endeavors to generate sustainable, biologically based solutions to ... where he held a variety of key roles including, ...
(Date:10/8/2015)... MANASSAS, Va. , Oct. 8, 2015  ATCC, ... announces today that it has been selected by The ... provide vital cell lines to academic, pharmaceutical, and biotechnology ... the United States , ... --> the United States , ...
(Date:10/8/2015)... 8, 2015 . ... --> Goldman Small Cap Research, a stock ... and microcap sectors, announced today that it has ... Biotech, Inc. (OTCQB - PMCB), a publicly traded, ... preparing treatments for cancer and diabetes. To view ...
Breaking Biology Technology:
... research, nanotechnology, farm waste management and produce storage have emerged ... Transfer Award. , ,Judges for the contest have winnowed the ... The $5,000 grand prize will be presented to the winner ... in Milwaukee. , ,The finalists are: , , GHD ...
... of Wisconsin and insert your card in an ATM in ... and your financial records. But if you find yourself in ... pay for an extensive series of tests because your medical ... doctor's office. , ,Information technology baffles users with ever-growing capabilities, ...
... a fast-food chain native to Wisconsin, is working with Madison ... old frying oil. Meanwhile, online extortionists are holding personal files ... entertainment has been rejected. , ,Original air date: Saturday, May ... , , Read more about the ...
Cached Biology Technology:
(Date:9/10/2015)... Report Details The global wearable ... previous expectations of revenues, consumer adoption and even technological ... which wearables begin to achieve that mass market acceptance ... reasons is the entrance of Apple to the SmartWatches ... but the overall size of the wearable technology market. ...
(Date:9/10/2015)... -- NXT-ID, Inc. (NASDAQ: NXTD ) ("NXT-ID" or the ... mobile commerce market, announces that the San ... will feature the Wocket® smart wallet for the first ... Touch Of Modern is an online retailer ... its 7.2 million members. Customers are affluent, urban influencers ...
(Date:9/8/2015)...   TREW Marketing and ... Marketing for Engineers TM : Content and Online Marketing ... findings about engineers, preferences for online content marketing and ... value and trust, and where they look for that ... grapple with how to best implement online and content ...
Breaking Biology News(10 mins):
... aging in mice has found that hormonal changes that occur ... established phenomenon: a low calorie diet can extend the lifespan ... achieve. We know that being lean rather than obese ... us that being lean from eating less, as opposed to ...
... Have Access to Critical Data with MobileCop(R) ... ... BIO-key International Inc.,(OTC Bulletin Board: BKYI), a leader in finger-based ... from the Baltimore (MD) Police Department ("BPD"),for additional licenses of BIO-key,s ...
... Swiss marine biologist and an Australian quantum physicist have found ... Australia, can see a world invisible to all other animals. ... shown that mantis shrimp not only have the ability to ... have optimal polarisation vision a first for any animal ...
Cached Biology News:
... is an automated solution for scheduling, performing ... testing for all pipettes and liquid handling ... balance Supports all gravimetric calibration ... 12650, ISO 9000, NCCLS and GLP requirements ...
... The QPatch sports 16-independent channels for automated ... used for screening against voltage-gated ion channels, ... as GABA channels. The four pipette ... throughput for ion channel drug discovery. ...
... Beads Sterile plating in just seconds ... and potentially hazardous ethanol flaming, More uniform ... ready-to-use sterile packaging: dispenser bottle for spreading 100 ... Fastest and easiest way to spread cells ...
... to successful cell culture or fermentation is ... the process, and make rapid adjustments in ... BioProfile Chemistry Analyzers provide immediate analysis of ... culture and fermentation media. BioProfile analyzers are ...
Biology Products: