Este trabalho foi financiado pelo NCI R01 CA120659 (RS).

1. A Matrigel-Based Assay formação do tubo para avaliar a atividade vasculogênica de células tumorais <ol><li> Preparação de células tumorais e células endoteliais microvasculares: células de tumor cerebral, como U87, células de melanoma B16F1, câncer de mama MDA-MB-435, e as células do cólon HCT116 foram cultivadas em DMEM suplementado com 10% FBS e penicilina / estreptomicina (todos da Invitrogen ). Células endoteliais microvasculares linhagem humana de células endoteliais (HMVECs) foram cultivados em meio EBM2 (Lonza) suplementado com 1 mg de hidrocortisona / ml e 1 ng / ml fator de crescimento epidérmico, 10% FBS e penicilina / estreptomicina. Células foram lavadas com PBS por três vezes e isolada com 0,05% de tripsina / EDTA (Invitrogen). Após a centrifugação com 2.000 rpm por 5 min, pellets de células foram lavadas novamente com PBS. Então, as células foram contadas peletizadas com um hemocitômetro. </li><li> Preparação Matrigel: Uma alíquota de fator de crescimento reduzida Matrigel (BD Bioscience) foi aquecido em temperatura ambiente. Antes completamente descongelado, foi transferido para gelo e seu líquido foi mantido no gelo por pelo menos 10 min. Em seguida, 50 ul de Matrigel foi banhado com placas de 96 poços em um nível horizontal que permite a Matrigel para distribuir uniformemente, e incubado por 30 min a 37 ° C. </li><li> Formação do tubo: Cells (1-2 x 10 4) foram re-suspensos com soro livre de DMEM para células tumorais ou EBM-2 para as células endoteliais, e colocado no topo da Matrigel. Se este ensaio foi utilizado para medir os efeitos de alguns agentes no desenvolvimento dos túbulos, esses agentes (estimuladores ou inibidores) foram adicionados ao meio isento de soro. Cada grupo continha condicional 4-6 poços. </li><li> Imagem e análise de dados: Após a incubação a 37 ° C durante a noite, cada poço foi analisado diretamente sob um microscópio. Se túbulos eram necessários para a fixação, 100 ul de solução salina 10% baseada em formol foi gentilmente adicionado e dez minutos depois, estava pronto para análise. Sob um microscópio com contraste de fase 10x, túbulos em cada campo foram fotografadas e uma média de túbulos 3-5 campos aleatórios em cada poço foi contado. </li></ol> 2. Resultados representativos: HMVECs foram utilizados como controle positivo, uma vez que estes túbulos foram desenvolvidos a partir clara corpos celulares alongadas que se unem para formar rede de polígono. Células B16F1, U87 e MDA-MB-435 desenvolvido túbulos vascular semelhantes aos formados por HMVECs, mas HCT116 células não (Figura 1). A formação do tubo celular dose-dependente foi testado em células U87. Como demonstrado na Figura 2, 10.000 células formadas túbulos descontinuado. Quando as células foram dobradas, uma sólida rede vascular foi comparável aos observados em HMVECs. Em contraste, as células menos de 5.000 não conseguiram formar um fenótipo vascular. <img src="/files/ftp_upload/3040/3040fig1.jpg" alt="Figura 1"/> Figura 1. Formação do tubo induzida por HMVECs, U87, MDA-MB-435, e B16F1 células, mas não HCT116 células. Todas as células (2 x 10 4) foram carregados em Matrigel e incubados durante a noite. Túbulos foram fotografadas usando contraste de fase. HMVECs foram utilizados como controle positivo. Um representante de 3-5 campos foi mostrado. Bar: 100 mm. <img src="/files/ftp_upload/3040/3040fig2.jpg" alt="Figura 2"/> Figura 2. U87 celular induzida por túbulos em um número de células-dependente maneira. Números diferentes de células U87, conforme indicado nos cantos foram usados ​​para a formação do tubo. HMVECs foram usados ​​para um controle 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>

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Para que este ensaio para o sucesso, a qualidade do Matrigel deve ser testada primeiro. Uma pequena amostra pode ser obtido a partir de BD Bioscience de pré-executar o teste usando HMVECs. Produtos de lote diferente pode exibir qualidades diferentes em que alguns lotes não fornecem uma condição ideal para a formação do tubo. Em segundo lugar, as bolhas devem ser evitados quando uma alíquota de Matrigel é carregado em placas de 96 poços, porque as bolhas podem interromper a formação de túbulos. Se pequenas bo...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </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> Do fator de crescimento reduzida 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 microscópio TS100 ECLIPSE </td><td> Nikon </td><td></td></tr></tbody></table>

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

Ao longo dos últimas décadas, um ensaio de formação do tubo utilizando fator de crescimento reduzida Matrigel foi tipicamente utilizados para demonstrar a atividade angiogênica das células endoteliais vasculares in vitro 1-5. No entanto, a evidência crescente recentemente demonstrou que este ensaio não se limita a testar o comportamento vascular para as células endoteliais. Em vez disso, ele também tem sido utilizado para testar a capacidade de um número de células tumorais para desenvolver um fenótipo vascular 6-8. Esta capacidade foi coerente com seu comportamento vasculogênica identificados em animais xenotransplanted, um processo conhecido como mimetismo vasculogênica (VM) 9. Há uma infinidade de provas que demonstrem que VM mediada por células tumorais desempenha um papel vital no desenvolvimento do tumor, independente de angiogênese da célula endotelial 6, 10-13. Por exemplo, as células tumorais foram encontradas para participar na formação do sangue perfundido canal, vascular em amostras de tecido de melanoma e glioblastoma pacientes 8, 10, 11. Aqui, descrevemos neste ensaio rede tubular como uma ferramenta útil na avaliação da atividade vasculogênica de células tumorais. Descobrimos que algumas linhagens de células tumorais, como o melanoma B16F1 células, as células U87 glioblastoma, e câncer de mama MDA-MB-435 células são capazes de formar túbulos vascular, mas algumas não, como câncer de cólon HCT116 células. Além disso, este fenótipo vascular é dependente número de células banhado no Matrigel. Portanto, este ensaio pode servir como poderoso utilitário para a tela o potencial vascular de uma variedade de tipos de células, incluindo células vasculares, as células tumorais, bem como outras 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

A formação do tubo de ensaio é usado para avaliar a atividade vascular das células tumorais.

A Matrigel-Based Assay formação do tubo para avaliar a atividade vasculogênica de células tumorais

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

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JoVE Journal

Biology

66.0K visualizações.  University of Massachusetts. A formação do tubo de ensaio é usado para avaliar a atividade vascular das células tumorais.

Vídeo: 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.

Um ensaio para medir a atividade do Escherichia coli Induzível 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 ...

Biologia

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.

Clonogênica Ensaio: células aderentes

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 de Cultura Primária células epiteliais trompa de Falópio

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

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 GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

Doseamento do atividade da quinase de 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

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.

Isolamento de células imunes de tumores primários

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.

Uma abordagem semi-quantitativa para avaliar formação de biofilmes usando o desenvolvimento da colônia enrugado

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.

Endotelial Tubo celular Formação ensaio para a<em&gt; In Vitro</em&gt; Estudo da angiogênese

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.

A Formação Ensaio macio 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

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.

Quantitative Reação de Imunofluorescência Indireta para medir a variação nos níveis de proteína no centrossomas

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.