SAS and MGA are supported by Project Grants, a Program Grant and Research Fellowships from the National Health and Medical Research Council of Australia (NHMRC), and by funds from the Operational Infrastructure Support Program provided by the Victorian Government, Australia. MMH has support from a Peter MacCallum Foundation Grant.

Source of IL-3 and Preparation of WEHI-3D-conditioned Medium
Note: The mouse granulocytic leukemia cell line WEHI-3D is cultured to generate a conditioned medium containing IL-3.
<ol>
	<li>Culture WEHI-3D in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), 10% fetal bovine serum (FBS), 1% long-life glutamine supplement, 50 &#956;g/ml gentamicin. Inoculate 5 &#215; 106 cells in the log phase of growth into 50 ml of fresh culture medium in a T175 cm3 tissue culture flas...

<ol>
	<li>Ferrara, N., Gerber, H. P., LeCouter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+VEGF+and+its+receptors.">The biology of VEGF and its receptors.</a> Nat Med. 9, 669-676 (2003).</li><li>Achen, M. G., Stacker, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor-D:signalling+mechanisms,+biology+and+clinical+relevance.">Vascular endothelial growth factor-D:signalling mechanisms, biology and clinical relevance.</a> Growth Factors. 5, 283-296 (2012).</li><li>Ferrara, N., Mass, R. D., Campa, C., Kim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+VEGF-A+to+treat+cancer+and+age-related+macular+degeneration.">Targeting VEGF-A to treat cancer and age-related macular degeneration.</a> Annu Rev Med. 58, 491-504 (2007).</li><li>Ferrara, N., Hillan, K. J., Gerber, H. P., Novotny, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+development+of+bevacizumab,+an+anti-VEGF+antibody+for+treating+cancer.">Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer.</a> Nat Rev Drug Discov. 3, 391-400 (2004).</li><li>Korpelainen, E. I., Alitalo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+angiogenesis+and+lymphangiogenesis.">Signaling angiogenesis and lymphangiogenesis.</a> Curr Opin Cell Biol. 10, 159-164 (1998).</li><li>Senger, D. R., 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=Tumour+cells+secrete+a+vascular+permeability+factor+that+promotes+accumulation+of+ascities+fluid.">Tumour cells secrete a vascular permeability factor that promotes accumulation of ascities fluid.</a> Science. 219, 983-985 (1983).</li><li>Joukov, V., 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=A+novel+vascular+endothelial+growth+factor,+VEGF-C,+is+a+ligand+for+the+Flt-4+(VEGFR-3)+and+KDR+(VEGFR-2)+receptor+tyrosine+kinases.">A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt-4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.</a> EMBO J. 15, 290-298 (1996).</li><li>Achen, M. G., 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=Vascular+endothelial+growth+factor+D+(VEGF-D)+is+a+ligand+for+the+tyrosine+kinases+VEGF+receptor+2+(Flk1)+and+VEGF+receptor+3+(Flt4).">Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).</a> Proc Natl Acad Sci USA. 95, 548-553 (1998).</li><li>Leppanen, V. M., 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=Structural+determinants+of+vascular+endothelial+growth+factor-D+receptor+binding+and+specificity.">Structural determinants of vascular endothelial growth factor-D receptor binding and specificity.</a> Blood. 117, 1507-1515 (2011).</li><li>Wise, L. M., 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=Vascular+endothelial+growth+factor+(VEGF)-like+protein+from+orf+virus+NZ2+binds+to+VEGFR2+and+neuropilin-1.">Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1.</a> Proc Natl Acad Sci USA. 96, 3071-3076 (1999).</li><li>Yamazaki, Y., Takani, K., Atoda, H., Morita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snake+venom+vascular+endothelial+growth+factors+(VEGFs)+exhibit+potent+activity+through+their+specific+recognition+of+KDR+(VEGF+receptor+2).">Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2).</a> J Biol Chem. 278, 51985-51988 (2003).</li><li>Stacker, S. A., Achen, M. G., Jussila, L., Baldwin, M. E., Alitalo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphangiogenesis+and+cancer+metastasis.">Lymphangiogenesis and cancer metastasis.</a> Nat Rev Cancer. 2, 573-583 (2002).</li><li>Stacker, S. A., 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=VEGF-D+promotes+the+metastatic+spread+of+tumor+cells+via+the+lymphatics.">VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.</a> Nat Med. 7, 186-191 (2001).</li><li>Skobe, M., 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=Induction+of+tumor+lymphangiogenesis+by+VEGF-C+promotes+breast+cancer+metastasis.">Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.</a> Nat Med. 7, 192-198 (2001).</li><li>Mandriota, S. 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=Vascular+endothelial+growth+factor-C-mediated+lymphangiogenesis+promotes+tumour+metastasis.">Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.</a> EMBO J. 20, 672-682 (2001).</li><li>Stacker, S. A., 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=Lymphangiogenesis+and+lymphatic+vessel+remodelling+in+cancer.">Lymphangiogenesis and lymphatic vessel remodelling in cancer.</a> Nat Rev Cancer. 14, 159-172 (2014).</li><li>Karnezis, T., 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=VEGF-D+promotes+tumor+metastasis+by+regulating+prostaglandins+produced+by+the+collecting+lymphatic+endothelium.">VEGF-D promotes tumor metastasis by regulating prostaglandins produced by the collecting lymphatic endothelium.</a> Cancer Cell. 21, 181-195 (2012).</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=Angiogenesis:+an+organizing+principle+for+drug+discovery.">Angiogenesis: an organizing principle for drug discovery.</a> Nat Rev Drug Discov. 6, 273-286 (2007).</li><li>Stacker, S. A., 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=Biosynthesis+of+vascular+endothelial+growth+factor-D+involves+proteolytic+processing+which+generates+non-covalent+homodimers.">Biosynthesis of vascular endothelial growth factor-D involves proteolytic processing which generates non-covalent homodimers.</a> J Biol Chem. 274, 32127-32136 (1999).</li><li>McColl, B. K., 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=Plasmin+activates+the+lymphangiogenic+growth+factors+VEGF-C+and+VEGF-D.">Plasmin activates the lymphangiogenic growth factors VEGF-C and VEGF-D.</a> J Exp Med. 198, 863-868 (2003).</li><li>Jaffe, E. A., Nachman, R. L., Becker, C. G., Minick, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+human+endothelial+cells+derived+from+umbilical+veins.+Identification+by+morphologic+and+immunologic+criteria.">Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.</a> J Clin Invest. 52, 2745-2756 (1973).</li><li>Shibuya, M., Claesson-Welsh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+by+VEGF+receptors+in+regulation+of+angiogenesis+and+lymphangiogenesis.">Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.</a> Exp Cell Res. 312, 549-560 (2006).</li><li>Pacifici, R. E., Thomason, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+tyrosine+kinase/cytokine+receptors+transmit+mitogenic+signals+in+response+to+ligand.">Hybrid tyrosine kinase/cytokine receptors transmit mitogenic signals in response to ligand.</a> J Biol Chem. 269, 1571-1574 (1994).</li><li>Stacker, S. A., 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=A+mutant+form+of+vascular+endothelial+growth+factor+(VEGF)+that+lacks+VEGF+receptor-2+activation+retains+the+ability+to+induce+vascular+permeability.">A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability.</a> J Biol Chem. 274, 34884-34892 (1999).</li><li>Davydova, N., Roufail, S., Streltsov, V. A., Stacker, S. A., Achen, M. 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+VD1+neutralizing+antibody+to+vascular+endothelial+growth+factor-D:+binding+epitope+and+relationship+to+receptor+binding.">The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding.</a> J Mol Biol. 407, 581-593 (2011).</li><li>Wise, L. M., 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=Viral+vascular+endothelial+growth+factors+vary+extensively+in+amino+acid+sequence,+receptor-binding+specificities,+and+the+ability+to+induce+vascular+permeability+yet+are+uniformly+active+mitogens.">Viral vascular endothelial growth factors vary extensively in amino acid sequence, receptor-binding specificities, and the ability to induce vascular permeability yet are uniformly active mitogens.</a> J Biol Chem. 278, 38004-38014 (2003).</li><li>Davydova, N., 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=Preparation+of+human+vascular+endothelial+growth+factor-D+for+structural+and+preclinical+therapeutic+studies.">Preparation of human vascular endothelial growth factor-D for structural and preclinical therapeutic studies.</a> Protein Expr. Purif. 82, 232-239 (2012).</li><li>Baldwin, M. E., 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=Multiple+forms+of+mouse+vascular+endothelial+growth+factor-D+are+generated+by+RNA+splicing+and+proteolysis.">Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis.</a> J. Biol. Chem. 276, 44307-44314 (2001).</li><li>Baldwin, M. E., 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=The+specificity+of+receptor+binding+by+vascular+endothelial+growth+factor-D+is+different+in+mouse+and+man.">The specificity of receptor binding by vascular endothelial growth factor-D is different in mouse and man.</a> J. Biol. Chem. 276, 19166-19171 (2001).</li><li>Makinen, T., 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=Isolated+lymphatic+endothelial+cells+transduce+growth,+survival+and+migratory+signals+via+the+VEGF-C/D+receptor+VEGFR-3.">Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3.</a> EMBOJ. 20, 4762-4773 (2001).</li><li>Achen, M. G., 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=Monoclonal+antibodies+to+vascular+endothelial+growth+factor-D+block+its+interactions+with+both+VEGF+receptor-2+and+VEGF+receptor-3.">Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.</a> Eur J Biochem. 267, 2505-2515 (2000).</li><li>Bamford, S., 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=The+COSMIC+(Catalogue+of+Somatic+Mutations+in+Cancer)+database+and+website.">The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website.</a> Br J Cancer. 91, 355-358 (2004).</li><li>Pleasance, E. D., 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=A+comprehensive+catalogue+of+somatic+mutations+from+a+human+cancer+genome.">A comprehensive catalogue of somatic mutations from a human cancer genome.</a> Nature. 463, 191-196 (2010).</li></ol>

Steven A. Stacker and Marc M. Achen are shareholders in Circadian Technologies Ltd., a company developing therapeutics by targeting the VEGF family of growth factors.

The assay described here relies on using cells of high viability, which are dependent on growth factors. Cells therefore need to be carefully cultured to ensure they are factor-dependent, and retain expression of the chimeric receptor. Ensuring that the medium is freshly made and not stored for an excessively long period and that WEHI-3D CM is highly active is important. Cells need to be thoroughly washed from IL-3 containing medium into the assay medium to ensure that no residual IL-3 contaminates the assay when exposin...

The vascular endothelial growth factor (VEGF) family of secreted protein growth factors and their cognate cell surface receptors is an important and diverse group of soluble ligands and membrane-embedded receptors, respectively that function in transducing signals across cellular membranes. They function mainly in endothelial cells but also in cells of epithelial origin and those of the immune system 1,2. Signaling pathways engaged by ligand-activated VEGF receptors (VEGFRs) are critical in major pathologies, such as age-related macular degeneration and cancer, and therapeutics targeting them are in frequent clinical use (e.g., the monoclonal antibody bevacizumab that targets VEGF-A) 3,4.
One of the complexities of the VEGF family is the diversity of soluble ligands present in nature (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF proteins encoded by the parapox virus family orf and snake venom VEGF, plus other inhibitory isoforms of VEGF-A)2.
These ligands interact with three members of the receptor tyrosine kinase family, namely VEGFR-1, VEGFR-2 and VEGFR-3. These receptors are variably expressed on different cell types but are often co-expressed on the surface of endothelial cells that line blood and lymphatic vessels of all sizes 5. VEGFR-2 can bind the mammalian ligands VEGF-A 6, VEGF-C 7 and VEGF-D 8,9 as well as orf virus VEGF10 and snake venom VEGF11. VEGFR-2 plays a major role in driving angiogenesis (the growth of new blood vessels from pre-existing vessels) in embryonic development, wound healing, cancer and eye diseases. In these contexts, ligands such as VEGF-A, -C and -D bind and activate the receptor on blood vascular endothelial cells12-15. On lymphatic endothelial cells, VEGFR-2 plays a role in lymphangiogenesis, the formation of new lymphatic vessels 16. VEGFR-2 can also promote dilation and expansion of major arteries and lymphatics in healthy tissues and disease 17. A complete understanding of VEGFR-2:ligand interactions is therefore important for the development of inhibitors for use in treating angiogenesis-dependent diseases18. While most isoforms of VEGF-A bind to VEGFR-2, proteolytic cleavage of VEGF-C and VEGF-D is required to release a fragment consisting of the VEGF-homology domain that exhibits high affinity binding to VEGFR-2 19,20.
We have developed a bioassay to monitor ligands of VEGFR-2 that is designed to circumvent the need for primary endothelial cells, which are technically difficult to passage, expensive to purchase and culture (requiring specialized medium) 21 and express multiple VEGFRs and associated co-receptors 22. Heterodimerization of VEGFR-2 with other VEGF receptors or co-receptors can cause unwanted complexity when aiming to study binary receptor-ligand interactions, evaluating activity attributable to a specific receptor, or assessing the effect of inhibitory reagents.23. The bioassay retains mobility of the relevant receptor in the cell membrane and allows evaluation of a ligand&#39;s ability to bind and cross-link the VEGFR-2 extracellular region.
The bioassay relies on the creation of a chimeric receptor in which the extracellular region of a VEGF receptor (in this case VEGFR-2) is fused to the transmembrane and intracellular regions of the erythropoietin receptor (EpoR), a member of the cytokine receptor family 8,24. This fusion protein is then expressed in the factor-dependent pro-B cell line Ba/F3, upon which stimulation with a ligand capable of binding and cross-linking the extracellular domain of the receptor causes activation of the cytoplasmic effector region, which is capable of transducing a survival signal via Janus kinases (JAKs) to promote cell survival and/or proliferation. In contrast, expression of full-length VEGFR-2 in the same cell type, and stimulation with ligand, does not promote cell survival and proliferation, indicating that the proximal signaling effectors of the VEGFR-2 pathway are not available in this cell type.
We have used the assay in a variety of contexts to explore binding of novel VEGFR-2 ligands 10,19,20,24-29. In combination with a VEGFR-3-EpoR-Ba/F3 assay, we have compared the relative activities of the VEGF-C and VEGF-D growth factors for binding and cross-linking VEGFR-2 and VEGFR-3 30. The assay has been used to characterize the inhibitory activity of neutralizing monoclonal antibodies to VEGFR-2 or VEGF-D, soluble VEGFR-2 trap and peptidomimetics targeting the VEGF family31. The assay was also used to show the ability of VEGFs from different orf virus strains to bind and cross-link VEGFR-2 prior to testing in primary endothelial cells 10,26. The assay is particularly useful for the rapid screening of mutants of VEGFs which can be quickly assessed for activity before they are introduced to the more laborious endothelial cell assays 25 or when assessing protocols for purifying growth factors 27.
The assay we describe is easy to perform, and the semi-quantitative version allows for quick determinations that are sometimes required when monitoring the production or purification of growth factors, antibodies or soluble receptor domains for other experiments. The ease of use of the assay makes it an ideal complement to further and more complete studies performed with primary endothelial cells derived from blood or lymphatic vessels from specific tissues or organ systems.

<table border="0" cellpadding="0" cellspacing="0" width="1110">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Trypan Blue</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:181px;">T8154</td>
			<td style="width:424px;">0.4% solution in PBS is used 1:1 with cell suspensions to measure viable cells. Hazard-may cause cancer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G418 Sulphate (Geneticin)</td>
			<td>Invivogen</td>
			<td>ant-gn-5</td>
			<td>Agent for selecting transfected eukaryotic cells. Hazard-may cause allergy or asthma symptoms or breathing difficluties.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3H-Thymidine</td>
			<td style="width:183px;">PerkinElmer</td>
			<td style="width:181px;">NET-027</td>
			<td>This radioactive nucleoside is incorporated into chromosomal DNA during mitosis. Hazard-radiation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vialight Plus Kit</td>
			<td>Lonza</td>
			<td>LT07-221</td>
			<td>Bioluminescent detection of cellular ATP to quantify viability, using ATP Monitoring Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prestoblue Cell Viability Reagent</td>
			<td>Invitrogen</td>
			<td>A13261</td>
			<td>Resazurin-based indicator of cell viability. Turns red in color in the reducing environment of the cell</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Nunc Minitray with Nunclon Delta Surface (72 well)</td>
			<td>Thermo Scientific</td>
			<td>136528</td>
			<td>Small microtitre plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">96 well Tissue Culture Plate</td>
			<td style="width:183px;">Falcon, Corning Inc.</td>
			<td style="width:181px;">353072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">DMEM (1X)</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">11965-92</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">GlutaMAX (100X)</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">35050-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Foetal Bovine Serum</td>
			<td style="width:183px;">Gibco</td>
			<td style="width:181px;">&#160;10099-141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell Harvester</td>
			<td style="width:183px;">Tomtec Life Sciences</td>
			<td style="width:181px;">N/A</td>
			<td>Tomtec Harvester, 96 Mach 3M Cell Harvester</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Scintillation Counter</td>
			<td style="width:183px;">LKB Wallac</td>
			<td style="width:181px;">1205</td>
			<td>LKB Wallac 1205 Betaplate Scintillation Counter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">UniFilter-96 GF/B</td>
			<td style="width:183px;">Perkin Elmer</td>
			<td>6005177</td>
			<td>White 96-well Barex Microplate with GF/B filterof 1 &#181;m poresize</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Gentamicin</td>
			<td style="width:183px;">Gibco, Life Technologies</td>
			<td style="width:181px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td style="width:183px;">Gibco, Life Technologies</td>
			<td style="width:181px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;width:323px;">0.22um pore cellulose acetate centrifuge tube filter unit</td>
			<td style="width:183px;">Costar, Corning Inc.</td>
			<td style="width:181px;">8160</td>
			<td>Centrifuge tube filters have a 0.22&#181;m pore CA membrane-containing filter unit within a 500&#181;l capacity polypropylene microcentrifuge tube.</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:16px;">Fluorescence Reader</td>
			<td style="width:183px;">BioTek</td>
			<td style="width:181px;">N/A</td>
			<td>BioTek Synergy 4 Hybrid Microplate Reader&#160;</td>
		</tr>
	</tbody>
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a simple bioassay for the evaluation of vascular endothelial growth factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

In this section, we show the results of an experiment demonstrating the essential features of a VEGFR-2-EpoR-Ba/F3 bioassay (see Figure 1 for principles of the assay). Other published studies demonstrate broader applications of the assay for alternative VEGFR-2 ligands, mutant VEGF molecules and inhibitory monoclonal antibodies 8,10,19,24-30.
The data presented here represent an assay in which the VEG...

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A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

The overall goal of this simple, cell-based bioassay is to detect, quantify, and monitor the activity of members of the vascular endothelial growth factor family of ligins. This method can answer key questions in vascular biology, virology, cancer, and opthamology, where VEG-FR family ligins radiate key processes in these diseases. The main advantage of this assay is it allows evaluation of VEG-FM family ligins in terms of their capacity to bind and cross link their different receptors without requiring specialized epithelial cells.Culture the IL3 secreting cell line WEHI-3D by pipetting five times ten to the sixth cells in the log face of growth into four to ten T175 flasks containing 50 millimeters of fresh culture medium. Incubate for seven days or until the cells have passed their log phase of growth by about 24 to 48 hours. Decant the fluid and cells from the flasks.And spin at 1, 000 Gs for 15 minutes to remove cells and cellular debris. Then remove the top 90%of supernaytent and filter it through a 0.22 micron filter unit. Aliquot the conditioned medium into one milliliter aliquots for assay preparation.50 milliliter aliquots for making culture medium to passage a factor dependent cell lines and 200 milliliter volumes for long term storage at 20 degree Celsius. Store the smaller volumes at 20 Celsius. Culture control BAF3 cells in culture media containing 10%WEHI-3D conditioned medium.and VegFR2-EPoR-BAF3 cells in the same media plus one milligram per milliliter G418. Passage cells at one to 15 dilutions from cells growing in log phase. Harvest control BAF3 or VegFR2VPoR-BAF3 cells for mid log phase cultures.By gently pipetting to remove the cells from the bottom of the flask centrifuge at 750 Gs for five minutes to recover cell pellet. Re-suspend the pellet in 10 milliliters of mouse tonicity phosphate buffered saline and centrifuge again at 750 Gs for five minutes. Repeat the wash two further times to remove medium containing IL3.Wash the cells once using media without DEHI-3D conditioned medium. It is important to wash away culture medium containing IL3 to ensure that no additional growth factors remain, which might generate a false positive result or high background activity. After centrifuging and discarding the supernayten re-suspend cells in the same IL3 free medium at a concentration of 7.4 times 10 to the fourth cells per milliliter.Finally mix equal volumes of trypan blue in PBS with a cell population and load into a hemacytometer. Count at least 100 cells. Cells that take up the dye are considered dead or dying.It is critical that cells used for the assay have high viability to ensure they are able to respond their assay conditions. Use a well calibrated P20 automated pipet and autoclaved tips to add 1, 000 cells in 13.5 microliters of IL deficient medium to the wells of a 72 well plate. Take care to mix the cell suspension during aliquoting to ensure cell settling by gravity does not bias cell concentration.Use a well calibrated P2 pipet to add 1.5 microliters of IL3 free media in triplicate to the control wells. Intrictery to mix. In the same manner add the 10%WEHI-3D conditioned medium and the 100 nanograms per milliliter VegFA no IL3 control to the desired wells.Finally add 1.5 microliters of the test samples in triplicate. Fill any unused wells of the microwell plate with sterile water, PBS or medium. Place water soaked tissue paper in a gas permeable container to create a humidified chamber that allows gas exchange.Incubate the assay plates in the chambers in a humidified atmosphere of 10%carbon dioxide for 16 hours. After 16 hours of incubation analyze the plates using a standard inverted phase microscope at 40 to 100x magnification. At this time it is possible to discriminate between positive and negative samples.Wells without support of growth factors or with medium alone will have reduced numbers of round cells as well as dead or dying cells and cellular debris. Whereas cells incubated with medium containing IL3 or ligin for VegFR2 are large and translucent and there is no or minimal evidence of granularity in the cell cytoplasm or cell debris in the culture. Add 10, 000 washed cells in 135 microliters of IL3 deficient medium to the wells of a standard 96 well plate.Use a calibrated P20 pipet to add 15 microliters of the test samples and controls to the wells. Incubate the mixture of cells growth factors and/or inhibitor agents as before for 48 hours. At the completion of the incubation period evaluate the number of viable cells in the wells using the methods outlined in the written portion of the protocol.Here are represented results of an assay in which the VEGF-R2 EPoR BAF3 bioassay is used to quantify the activity of the three recumbent human ligins with specificity for VEGF-R2. VEGF-A165, VEGF-C, and VEGF-D. Activity was quantified in the presence of bevacizumab, an inhibitory monoclonal antibody to VEGF-A165 or in the presence of trastuzumab, a non neutralizing antibody.Data have been normalized versus the response to VEGF-A165 alone. As expected bevacizumab blocked the effect of VEGF-A165 in the assay but had no effect on the activity of VEGF-C and VEGF-D. Cells exposed to IL3 were highly viable.Whereas those exposed to no additional growth factor show poor viability. When attempting this procedure it's important to bare in mind that they're a range of controlling agents in conditioned media that need to be generated prior to the assay. This technique paved the way for resurgence in vascular biology, virology, cancer and opthomology to explore the role of VEG-FM in ligins.After watching this video you should have a good understanding how to prepare the bioassay cells, to run the bioassay and to conduct the quantitative or semiquantitative analyses of VEG-FM ligin activity.

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9.6K Görüntüleme.  Peter MacCallum Cancer Centre. The overall goal of this simple, cell-based bioassay is to detect, quantify, and monitor the activity of members of the vascular endothelial growth factor family of ligins. This method can answer key questions in vascular biology, virology, cancer, and opthamology, where VEG-FR family ligins radiate key processes in these diseases. The main advantage of this assay is it allows evaluation of VEG-FM family ligins in terms of their capacity to bind and cross link their different receptors withou...