Name: a matrigel-based tube formation assay to assess the vasculogenic activity of tumor cells
Uploaded: 2011-09-07T13:00:00
Description: Watch this Scientific Journal Video about A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells at JoVE.com

Questo lavoro è stato sostenuto dal NCI R01 CA120659 (RS).

1. Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali <ol><li> Preparazione delle cellule tumorali e cellule endoteliali: le cellule tumorali del cervello, come le cellule U87, B16F1 cellule di melanoma, cancro al seno MDA-MB-435, e le cellule del colon HCT116 sono state coltivate in DMEM supplementato con 10% FBS e la penicillina / streptomicina (tutti da Invitrogen ). Delle cellule endoteliali linea di cellule endoteliali microvascolari (HMVECs) sono state coltivate...

<ol>
	<li>Shao, R., Guo, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvascular+endothelial+cells+immortalized+with+human+telomerase+catalytic+protein:+a+model+for+the+study+of+in+vitro+angiogenesis.">Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis.</a> Biochemical &#38; Biophysical Research Communications. 321, 788-794 (2004).</li><li>Ribeiro, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemostatic+properties+of+the+SV-40+transfected+human+microvascular+endothelial+cell+line+(HMEC-1).+A+representative+in+vitro+model+for+microvascular+endothelium.">Hemostatic properties of the SV-40 transfected human microvascular endothelial cell line (HMEC-1). A representative in vitro model for microvascular endothelium.</a> Thromb Res. 79, 153-1561 (1995).</li><li>Ades, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMEC-1:+establishment+of+an+immortalized+human+microvascular+endothelial+cell+line.">HMEC-1: establishment of an immortalized human microvascular endothelial cell line.</a> J Invest Dermatol. 99, 683-690 (1992).</li><li>Shao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquired+expression+of+periostin+by+human+breast+cancers+promotes+tumor+angiogenesis+through+up-regulation+of+vascular+endothelial+growth+factor+receptor+2+expression.">Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression.</a> Molecular &#38; Cellular Biology. 24, 3992-4003 (2004).</li><li>Shao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YKL-40,+a+secreted+glycoprotein,+promotes+tumor+angiogenesis.">YKL-40, a secreted glycoprotein, promotes tumor angiogenesis.</a> Oncogene. 28, 4456-4468 (2009).</li><li>Basu, G. D. <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+role+for+cyclooxygenase-2+in+regulating+vascular+channel+formation+by+human+breast+cancer+cells.">A novel role for cyclooxygenase-2 in regulating vascular channel formation by human breast cancer cells.</a> Breast Cancer Research. 8, R69-R69 (2006).</li><li>Scavelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenic+mimicry+by+bone+marrow+macrophages+in+patients+with+multiple+myeloma.">Vasculogenic mimicry by bone marrow macrophages in patients with multiple myeloma.</a> Oncogene. 27, 663-674 (2008).</li><li>El Hallani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+alternative+mechanism+in+glioblastoma+vascularization:+tubular+vasculogenic+mimicry.">A new alternative mechanism in glioblastoma vascularization: tubular vasculogenic mimicry.</a> Brain. 133, 973-982 (2010).</li><li>Maniotis, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+channel+formation+by+human+melanoma+cells+in+vivo+and+in+vitro:+vasculogenic+mimicry.">Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry.</a> American Journal of Pathology. 155, 739-752 (1999).</li><li>Hendrix, M. J., Seftor, E. A., Hess, A. R., Seftor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenic+mimicry+and+tumour-cell+plasticity:+lessons+from+melanoma.">Vasculogenic mimicry and tumour-cell plasticity: lessons from melanoma.</a> Nature Reviews Cancer. 3, 411-421 (2003).</li><li>Folberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+plasticity+in+uveal+melanoma:+microenvironment+directed+dampening+of+the+invasive+and+metastatic+genotype+and+phenotype+accompanies+the+generation+of+vasculogenic+mimicry+patterns.">Tumor cell plasticity in uveal melanoma: microenvironment directed dampening of the invasive and metastatic genotype and phenotype accompanies the generation of vasculogenic mimicry patterns.</a> American Journal of Pathology. 169, 1376-1389 (2006).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate-specific+membrane+antigen+directed+selective+thrombotic+infarction+of+tumors.">Prostate-specific membrane antigen directed selective thrombotic infarction of tumors.</a> Cancer Research. 62, 5470-5475 (2002).</li><li>Sood, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+significance+of+tumor+cell-lined+vasculature+in+ovarian+carcinoma:+implications+for+anti-vasculogenic+therapy.">The clinical significance of tumor cell-lined vasculature in ovarian carcinoma: implications for anti-vasculogenic therapy.</a> Cancer Biology &#38; Therapy. 1, 661-664 (2002).</li><li>Shirakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+in+vasculogenic+mimicry+and+angiogenesis+of+inflammatory+breast+cancer+xenograft.">Hemodynamics in vasculogenic mimicry and angiogenesis of inflammatory breast cancer xenograft.</a> Cancer Research. 62, 560-566 (2002).</li><li>Ruf, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+role+of+tissue+factor+pathway+inhibitors+1+and+2+in+melanoma+vasculogenic+mimicry.">Differential role of tissue factor pathway inhibitors 1 and 2 in melanoma vasculogenic mimicry.</a> Cancer Research. 63, 5381-5389 (2003).</li><li>Seftor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+interactions+of+laminin+5+gamma2+chain,+matrix+metalloproteinase-2,+and+membrane+type-1-matrix/metalloproteinase+are+required+for+mimicry+of+embryonic+vasculogenesis+by+aggressive+melanoma.">Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.</a> Cancer Research. 61, 6322-6327 (2001).</li><li>Shirakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasculogenic+mimicry+and+pseudo-comedo+formation+in+breast+cancer.">Vasculogenic mimicry and pseudo-comedo formation in breast cancer.</a> International Journal of Cancer. 99, 821-828 (2002).</li></ol>

Perché questo test per avere successo, la qualità del Matrigel deve essere testato prima. Un piccolo campione possono essere ottenute presso Bioscience BD per pre-eseguire il test utilizzando HMVECs. Prodotti lotti diversi possono visualizzare qualità dissimili in cui alcuni lotti non forniscono una condizione ottimale per la formazione del tubo. In secondo luogo, tutte le bolle dovrebbe essere evitato quando un&#39;aliquota del Matrigel viene caricato in piastre a 96 pozzetti, perché le bolle possono interrompere l...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11995 </td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16000-044 </td></tr><tr><td> Fattore di crescita ridotto Matrigel </td><td> BD Bioscience </td><td> 47743-720 </td></tr><tr><td> EBM2 kit </td><td> Lonza </td><td> CC-3156 </td></tr><tr><td> Nikon ECLIPSE TS100 microscopio </td><td> Nikon </td><td></td></tr></tbody></table>

a matrigel-based tube formation assay to assess the vasculogenic activity of tumor cells

Nel corso degli ultimi decenni, un saggio di formazione del tubo utilizzando il fattore di crescita-è stato ridotto Matrigel tipicamente impiegato per dimostrare l&#39;attività angiogenica delle cellule dell&#39;endotelio vascolare in vitro 1-5. Tuttavia, l&#39;evidenza di recente crescita ha dimostrato che questo saggio non si limita a testare il comportamento per le cellule endoteliali vascolari. Invece, è stato anche utilizzato per testare la capacità di un certo numero di cellule tumorali di sviluppare un fenotipo vascolare 6-8. Questa capacità è stata coerente con il loro comportamento vascolare identificato in animali xenotransplanted, un processo noto come mimetismo vascolare (VM) 9. C&#39;è una moltitudine di prove che dimostrano che cellule tumorali mediata VM svolge un ruolo fondamentale nello sviluppo del tumore, indipendentemente angiogenesi delle cellule endoteliali 6, 10-13. Per esempio, le cellule tumorali sono stati trovati a partecipare al perfuso sangue, la formazione di canali vascolari in campioni di tessuto da pazienti affetti da melanoma e glioblastoma 8, 10, 11. Qui, abbiamo descritto questa analisi tubolare rete come uno strumento utile nella valutazione dell&#39;attività vascolare delle cellule tumorali. Abbiamo scoperto che alcune linee cellulari tumorali come il melanoma B16F1 cellule, le cellule di glioblastoma U87, e il cancro al seno MDA-MB-435 cellule sono in grado di formare tubuli vascolare, ma alcuni non lo fanno come il cancro del colon HCT116 cellule. Inoltre, questo fenotipo vascolare dipende dal numero di cellule placcato sul Matrigel. Pertanto, questo test può servire come potente utility per lo screening del potenziale vascolare di una varietà di tipi cellulari tra cui le cellule vascolari, le cellule tumorali oltre che altre cellule.

Watch this Scientific Journal Video about A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells at JoVE.com

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Un test formazione del tubo viene usato per valutare l&#39;attività vascolare delle cellule tumorali.

Un Matrigel-Based Formazione Assay tubo per valutare l&#39;attività vascolare delle cellule tumorali

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Ricerca

Biologia

Clonogenic Assay: Adherent Cells

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Ex Cultura Vivo di cellule primarie umane tube di Falloppio epiteliale

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Assaying the Kinase Activity of LRRK2 in vitro

Assaying the Kinase Activity of LRRK2 in vitro

Saggiare l&#39;attività chinasi di LRRK2 In vitro</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Isolation of Immune Cells from Primary Tumors

Isolamento di cellule immunitarie di tumori primari

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Un approccio semi-quantitativo per valutare la formazione di biofilm mediante lo sviluppo spiegazzato Colony

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

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

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

Endoteliali Tubo cellulare saggio Formazione per la<em&gt; In Vitro</em&gt; Studio di Angiogenesi

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

The Soft Agar Colony Formation Assay

Il saggio Formazione morbida Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ​​ad centrosomi

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

In vitro misurare phosphatidylethanolamine metiltransferasi attività

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from ...