Name: a simple bioassay for the evaluation of vascular endothelial growth factors
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Description: Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

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>

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>
</table>

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...

Watch this Scientific Journal Video about A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors at JoVE.com

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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Research

JoVE Journal

Biology

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Demonstrating a Linear Relationship Between Vascular Endothelial Growth Factor and Luteinizing Hormone in Kidney Cortex Extracts

Vascular endothelial growth factor (VEGF) helps to control angiogenesis and vascular permeability in the kidney. Renal disorders, such as diabetic nephropathy, are associated with VEGF dysregulation in the kidney. The factors that govern VEGF under physiologic conditions in the kidney are not well-understood. Luteinizing hormone (LH), a pro-angiogenic hormone, helps regulate physiologic VEGF expression in reproductive organs. Given that LH receptors are found in the kidney, we, at Zietchick Research Institute, hypothesized here that LH also helps regulate VEGF expression in the kidney as well. To provide evidence, we aimed to show that LH levels are able to predict VEGF levels in the mammalian kidney. Most VEGF-related investigations involving the kidney have used lower order mammals as models (i.e., rodents and rabbits). To translate this work to the human body, it was decided to examine the relationship between VEGF and LH in higher order mammals (i.e., bovine and porcine models). This protocol uses the total protein lysate from the kidney cortex. Keys to this method&#39;s success include procurement of kidneys from slaughterhouse animals immediately after death as well as normalization of analyte levels (in the kidney extract) by total protein. This study successfully demonstrates a significant linear relationship between LH and VEGF in both bovine and porcine kidneys. The results are reproducible in two different species. The study provides supporting evidence that the use of kidney extracts from cows and pigs are an excellent, economical, and abundant resource for the study of renal physiology, particularly for examining the correlation between VEGF and other analytes.

Presented here is a protocol for utilizing a cortical kidney extract preparation and total protein normalization to demonstrate the correlation between vascular endothelial growth factor and luteinizing hormone in the mammalian kidney.