Este trabajo fue financiado por el NCI R01 CA120659 (RS).

1. A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica <ol><li> Preparación de las células tumorales y las células endoteliales microvasculares: las células de tumores cerebrales, tales como U87 células, las células del melanoma B16F1, el cáncer de mama MDA-MB-435, y las células del colon HCT116 fueron cultivadas en DMEM suplementado con FBS al 10% y penicilina / estreptomicina (todos de Invitrogen ). Línea de células endoteliales humanas células endoteliales microvasculares (HMVECs) se cultivaron en medio EBM2 (Lonza), complementado con 1 mg / ml de hidrocortisona y 1 ng / ml de factor de crecimiento epidérmico, el 10% de SFB y penicilina / estreptomicina. Las células fueron lavadas tres veces con PBS y distante con el 0,05% de tripsina / EDTA (Invitrogen). Después de la centrifugación a 2.000 rpm durante 5 min, gránulos de las células se lavaron de nuevo con PBS. Luego, las células sedimentadas se contó con un hemocitómetro. </li><li> Preparación Matrigel: una alícuota de factor de crecimiento reducido Matrigel (BD Bioscience) se calentó a temperatura ambiente. Antes de que se están descongelando, fue trasladado en hielo y el líquido se mantuvo en hielo durante al menos 10 min. De 50 l de Matrigel se sembró a 96 y placas a un nivel horizontal que permite que el Matrigel para distribuir uniformemente, y se incubó durante 30 min a 37 ° C. </li><li> La formación del tubo: las células (1-2 x 10 4) se volvió a suspender con suero libre de DMEM para las células tumorales o EBM-2 para las células endoteliales, y se carga en la parte superior de la Matrigel. Si este ensayo se utilizó para medir los efectos de algunos agentes en el desarrollo de los túbulos, estos agentes (estimuladores o inhibidores) se añadieron al medio libre de suero. Cada grupo condicional contenida 4-6 pozos. </li><li> Imagen y análisis de datos: Después de la incubación a 37 ° C durante la noche, cada uno así se analizan directamente bajo un microscopio. Si los túbulos se necesitaban para la fijación, 100 l de formalina salina al 10% basado en solución se añadió con suavidad y diez minutos más tarde, estaba listo para su análisis. Bajo un microscopio con contraste de fase 10x, los túbulos en cada campo fueron fotografiadas y un promedio de túbulos 3-5 campos al azar en cada pozo se había contado. </li></ol> 2. Los resultados representativos: HMVECs se utilizaron como control positivo, ya que estos tubos se desarrollaron a partir clara cuerpos celulares alargados que conectan a la red de forma poligonal. Células B16F1, U87, y MDA-MB-435 desarrollado túbulos vasculares similares a las formadas por HMVECs, pero HCT116 células no (Figura 1). Una célula dependiente de la dosis la formación del tubo se ha probado en células U87. Como se demuestra en la figura 2, 10000 células formadas túbulos interrumpido. Una vez que las células se duplicó, de una red vascular sólido es comparable a los observados en HMVECs. En contraste, las células de menos de 5.000 no lograron formar un fenotipo vascular. <img src="/files/ftp_upload/3040/3040fig1.jpg" alt="Figura 1"/> Figura 1. Formación del tubo inducida por HMVECs, U87, MDA-MB-435, y las células B16F1, pero no las células HCT116. Todas las células (2 x 10 4) fueron cargados en Matrigel y se incubaron durante la noche. Túbulos estaban utilizando imágenes de contraste de fase. HMVECs se utilizaron como control positivo. Un representante de 3-5 campos se muestran. Bar: 100 micras. <img src="/files/ftp_upload/3040/3040fig2.jpg" alt="Figura 2"/> Figura 2. U87 celular inducida por los túbulos, en un número de celular dependiente de forma. Diferentes números de células U87 como se indica en las esquinas se utilizan para la formación del tubo. HMVECs fueron utilizados para un control positivo. Bar: 100 mm.

<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>

No hay conflictos de interés declarado.

A fin de que este ensayo tenga éxito, la calidad de Matrigel debe ser probado por primera vez. Una pequeña muestra se puede obtener de BD Bioscience de pre-ejecución del ensayo utilizando HMVECs. Diferentes productos por lotes puede mostrar cualidades diferentes en los que algunos lotes no ofrecen condiciones óptimas para la formación del tubo. En segundo lugar, las burbujas deben ser evitados cuando una parte alícuota de Matrigel se carga en placas de 96 pozos, porque las burbujas pueden alterar la formación de ...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </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> El factor de crecimiento reducido 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

Durante las últimas décadas, un ensayo de la formación del tubo con el factor de crecimiento reducido Matrigel ha sido generalmente empleado para demostrar la actividad angiogénica de las células endoteliales vasculares in vitro 1-5. Sin embargo, evidencia reciente ha demostrado cada vez que este ensayo no se limita a probar el comportamiento de las células endoteliales vasculares. Por el contrario, también se ha utilizado para poner a prueba la capacidad de un número de células tumorales a desarrollar un fenotipo vascular 6-8. Esta capacidad fue consistente con su comportamiento vasculogénica identificado en animales xenotransplantado, un proceso conocido como vasculogénica mímica (VM) 9. Hay una gran cantidad de evidencias que demuestran que las células tumorales mediada por VM juega un papel vital en el desarrollo del tumor, con independencia de la angiogénesis de las células endoteliales 6, 10-13. Por ejemplo, las células tumorales se encuentran para participar en la sangre perfunde la formación, el canal vascular en muestras de tejido de pacientes con melanoma y glioblastoma 8, 10, 11. En este sentido, este ensayo se describe la red tubular como una herramienta útil en la evaluación de la actividad vasculogénica de las células tumorales. Hemos encontrado que algunas líneas celulares tumorales como el melanoma B16F1 células, las células de glioblastoma U87, y el cáncer de mama MDA-MB-435 células son capaces de formar túbulos vasculares, pero algunos no lo hacen, como el cáncer de colon HCT116 células. Además, este fenotipo vascular depende de el número de células chapada en el Matrigel. Por lo tanto, este ensayo puede servir de gran utilidad para detectar el potencial vascular de una variedad de tipos de células incluyendo las células vasculares, las células tumorales, así como otras células.

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 ensayo de la formación del tubo se utiliza para evaluar la actividad vascular de las células tumorales.

A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica

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

a-matrigel-based-tube-formation-assay-to-assess-vasculogenic-activity

Research

JoVE Journal

Biology

65.8K vistas.  University of Massachusetts. Un ensayo de la formación del tubo se utiliza para evaluar la actividad vascular de las células tumorales.

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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine 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 acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

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

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un ensayo para medir la actividad de Escherichia coli Inducible 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 ...

Investigación

Biología

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Ensayo clonigénicas: las células adherentes

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

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 uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

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 ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

El aislamiento de las células inmunes de tumores primarios

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

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

Un enfoque semi-cuantitativo para evaluar la formación de biopelículas Con el desarrollo de colonias arrugada

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

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

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

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

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

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

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

El ensayo de formación de colonias en agar blando

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

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Cuantitativa inmunofluorescencia Ensayo para medir la variación en los niveles de proteína en centrosomas

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

Phosphatidylethanolamine methyltransferases are biosynthetic enzymes that catalyze the transfer of one or more methyl group(s) from S-adenosyl-L-methionine onto phosphatidylethanolamine, monomethyl-phosphatidylethanolamine, or dimethyl-phosphatidylethanolamine to give either monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine or phosphatidylcholine. These enzymes are ubiquitous in animal cells, fungi, and are also found in approximately 10% of bacteria. They fulfill various important functions in cell physiology beyond their direct role in lipid metabolism such as in insulin resistance, diabetes, atherosclerosis, cell growth, or virulence. The present manuscript reports on a simple cell-free enzymatic assay that measures the transfer of tritiated methyl group(s) from S-[Methyl-3H]adenosyl-L-methionine onto phosphatidylethanolamine using whole cell extracts as an enzyme source. The resulting methylated forms of phosphatidylethanolamine are hydrophobic and thus, can be separated from water soluble S-[Methyl-3H]adenosyl-L-methionine by organic extraction. This assay can potentially be applied to any other cell types and used to test inhibitors/drugs specific to a phosphatidylethanolamine methyltransferase of interest without the need to purify the enzyme.

In Vitro Assay to Measure Phosphatidylethanolamine Methyltransferase Activity

The present report describes an in vitro enzymatic assay to measure phosphatidylethanolamine methyltransferase activity using Leishmania cell extracts. This assay is based on the transfer of a radioactive methyl group from S-[Methyl-3H]adenosyl-L-methionine onto endogenous phosphatidylethanolamine.

Ensayo in vitro para medir Fosfatidiletanolamina metiltransferasa actividad

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina Microinyección en pez cebra para modelar la lesión renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...