This research was supported in part by the Intramural Research Program of the National Cancer Institute at the National Institutes of Health, and by NCI grant UA5CA152907.

1. Collection of Conditioned Media to Test for Angiogenic Potential
<ol>
	<li>Grow primary or immortalized cells to be tested for angiogenic or anti-angiogenic potential in native or low serum media and collect the conditioned media.
	<ol>
		<li>Alternatively, use conditioned media immediately, or aliquoted and stored at -80 &#176;C for several months. Use nonconditioned native or low serum media as a negative control, and use non-conditioned complete growth media (10% FBS, or appropriate concentration) as a positive c...

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438, 932-936 (1038).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146, 873-887 (2011).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438, 937-945 (2005).</li><li>Bouis, D., Kusumanto, Y., Meijer, C., Mulder, N. H., Hospers, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+pro-+and+anti-angiogenic+factors+as+targets+of+clinical+intervention.">A review on pro- and anti-angiogenic factors as targets of clinical intervention.</a> Pharmacological research : the official journal of the Italian Pharmacological Society. 53, 89-103 (2006).</li><li>Ausprunk, D. H., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+proliferation+of+endothelial+cells+in+preformed+and+newly+formed+blood+vessels+during+tumor+angiogenesis.">Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis.</a> Microvascular research. 14, 53-65 (1977).</li><li>Chung, A. S., Lee, J., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tumour+vasculature:+insights+from+physiological+angiogenesis.">Targeting the tumour vasculature: insights from physiological angiogenesis.</a> Nature reviews. Cancer. 10, 505-514 (2010).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annual review of cell and developmental biology. 27, 563-584 (2011).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England journal of medicine. 358, 2039-2049 (2008).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England journal of medicine. 285, 1182-1186 (1056).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> The Journal of cell biology. 107, 1589-1598 (1988).</li><li>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12, 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature protocols. 5, 628-635 (2010).</li><li>Walter-Yohrling, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+endothelial+cell+lines+as+models+of+tumor+endothelial+cells.">Murine endothelial cell lines as models of tumor endothelial cells.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 10, 2179-2189 (2004).</li><li>Connell, K. A., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+lymphoid+endothelial+cell+line+immortalized+by+simian+virus+40+binds+lymphocytes+and+retains+functional+characteristics+of+normal+endothelial+cells.">A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.</a> Journal of immunology. , 144-521 (1950).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+contribution:+Angiogenesis+Analyzer.">ImageJ contribution: Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li></ol>

The authors have nothing to disclose.

Angiogenesis is involved in both physiological and pathological processes. Studying mechanisms involved in angiogenesis requires the use of assays that recapitulate the major steps in angiogenesis. The endothelial cell tube formation assay offers several advantages over other assays. It is easy to set-up, is relatively inexpensive, produces tubules within hours, and is quantifiable. Further, it can be completed in 24- or 96-well plates, and thus can be used for high throughput screening to identify factors that stimulate...

Angiogenesis, the development of new blood vessels from preexisting vessels, is vital for a variety of processes including organ growth, embryonic development, and wound healing1-3. Newly developed blood vessels, lined by endothelial cells, supply oxygen and nutrients to tissues, promote immune surveillance by hematopoietic cells and remove waste products2,4. Angiogenesis is of key importance during embryonic and fetal development. However, this process remains dormant in the adult except during times of wound healing, skeletal growth, pregnancy or during the menstrual cycle1-3.
Over the last two decades, key molecular mechanisms that regulate angiogenesis have begun to emerge. Angiogenesis is a tightly regulated event, balanced by pro and antiangiogenic signals including integrins, chemokines, angiopoietins, oxygen sensing agents, junctional molecules and endogenous inhibitors5. Once proangiogenic signals such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and epidermal growth factor (EGF) activate endothelial cell receptors the endothelial cells release proteases to degrade the basement membrane. The endothelial cells then proliferate and migrate, forming sprouts at a rate of several millimeters per day6,7.
Angiogenesis is associated with various pathological conditions including cancer, psoriasis, diabetic retinopathy, arthritis, asthma, autoimmune disorders, infectious diseases, and atherosclerosis8-10. Due to the importance of angiogenesis in various diseases, understanding the genes and pathways that regulate this process is critical to the design of better therapeutics.
The tube formation assay is a rapid and quantitative method for determining genes or pathways involved in angiogenesis. First described in 1988, the principles underlying this assay are that endothelial cells retain the ability to divide and migrate rapidly in response to angiogenic signals11-13. Further, endothelial cells are induced to differentiate and form tube-like structures when cultured on a matrix of basement membrane extract (BME). These tubes contain a lumen surrounded by endothelial cells linked together through junctional complexes. Tube formation occurs quickly with most tubes forming in this assay within 2-6 hr depending on quantity and type of angiogenic stimuli.
Several types of endothelial cells can be used for this assay including both primary cells and immortalized cell lines14,15.&#160;The cell line used for this article was mouse 3B-11, but the same methodology can be applied with other endothelial cell lines such as SVEC4-10 (mouse) or primary endothelial cells such as HUVEC (human) cells. Depending on which cell line is used and whether the endothelial cells are transformed or non-transformed, optimization will need to be conducted to identify the ideal time needed for proper tube formation.

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			<td height="42" rowspan="2" style="height:42px;width:339px;">Item</td>
			<td rowspan="2" style="width:189px;">Manufacturer</td>
			<td rowspan="2" style="width:124px;">Catalog #</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="93">
			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="169">
			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="55">
			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25300-054</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="74">
			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">14190-144</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Fetal Bovine Serum</td>
			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
		</tr>
	</tbody>
</table>

endothelial cell tube formation assay for the in vitro study of angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Mouse 3B-11 endothelial cells were seeded on solidified reduced growth factor BME &#8211; in this assay, the product Matrigel was used - and followed over time. As shown in Figure 1, angiogenic factors secreted by either mouse keratinocytes or fibroblasts are capable of inducing tube formation over time. Endothelial cells migrate and begin to form small branches within 1-2 hr of plating. Maximum tube formation was reached by 4-6 hr using conditioned media previously obtained from keratinocytes. By 24 hr,...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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58.8K visualizações.  American University.  O ensaio de formao de tubo é um método rápido, quantificável para medir a angiogénese in vitro. As células endoteliais são combinados com meios condicionados e semeadas em extracto de membrana basal. A formação do tubo ocorre dentro de poucas horas e túbulos recém-formados facilmente quantificada. 