Este trabalho foi financiado por uma bolsa da New Innovator Award Program Director do NIH (ED). AA foi apoiado em parte por prêmios de graduação do Instituto Nacional do Câncer e da Fundação Nacional de Ciência.

1. Fazer os vetores virais As sequências de codificação para o gene de interesse, bem como os controlos positivos e negativos, são inseridos em pBABEpuro por métodos tradicionais de clonagem (amplificação por PCR, digestão de restrição e de ligação). Existem quatro locais de restrição no vector em que o DNA pode ser inserido: BamHI, SnaBI, EcoRI e Sall. <ol><li> Prepare plasmídeo DNA transfecção-grade a partir de células competentes, segundo o kit plasmídeo midi QIAGEN. </li><li> Medir a concentração de DNA usando um espectrofotômetro NanoDrop2000. </li></ol> 2. Retrovirus Produção A linha celular de empacotamento Plat-E vai ser utilizado para produzir retrovírus ecotrópico que permitirá obter o ADNc de interesse a células NIH 3T3. <ol><li> Estabelecer Plat-E culturas de células congeladas <ol><li> Rapidamente descongelar células Plat-E de um ° C banho de água 37 e transferir para um tubo de 15 ml. Lentamente, adicionar 9 ml de meio de Plat-E (DuMeio Eagle Modificado de lbecco (DMEM) + 10% de Soro Fetal Bovino (FBS) + penicilina e estreptomicina). </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Re-suspender as células com 10 ml de Plat-E e meio de transferência para uma placa de cultura de 10 centímetros. </li><li> Incubar as células em uma temperatura de 37 ° C, 5% de CO2 até que eles são de 80-90% confluentes (cerca de 2 dias). </li></ol></li><li> Splitting celular <ol><li> Aspirar o meio e lave as células uma vez com PBS. </li><li> Adicionar 2 ml de 0,05% de tripsina-EDTA e incubar durante 1 minuto à temperatura ambiente. </li><li> Retire as células por finger tapping, adicione 10 ml de meio Plat-E, e transferir a suspensão de células para um tubo de 15 ml. </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Ressuspender as células com 10 ml de meio Plat-E e semear-los em novas placas a 1: 4-1: 6 diluições. </li></ol></li><li> Plat-E Sementeira e Transfection <ol><li> Semente de 2 x 10 6células por 10 cm prato de cultura, utilizando meio de Plat-E sem quaisquer antibióticos. </li><li> Incubar as células S / N em uma temperatura de 37 ° C, 5% de CO2. </li><li> No dia seguinte, transferir 300 ul de Opti-MEM em 1,5 ml de tubo de microcentrífuga. </li><li> Adicionar 27 ul de polietilenimina (PEI), um reagente de transfecção de baixo custo 12, o tubo preparado com Opti-MEM. Misturar suavemente batendo por dedo e incubar durante 5 min à TA. </li><li> Adicionar 9 ug de ADN plasmídeo de transfecção de grau para o tubo de Opti-MEM / PEI, misturar suavemente em vórtex e incubar durante 15 min à temperatura ambiente. </li><li> Adicionar o ADN / PEI gota a gota complexo no prato Plat-E, e incubar O / N a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, aspirar o meio contendo o reagente de transfecção e adicione 10 ml de meio Plat-E fresco (sem antibióticos). </li><li> Retorne as células para a incubadora. </li></ol></li></ol> 3. As células NIH 3T3 e Infecção <ol><li> Estabelecer NIHCultures 3T3 e propagação <ol><li> Rapidamente descongelar células NIH 3T3 numa ° C num banho de água a 37 e transferir para um tubo de 15 ml. Adiciona-se lentamente 9 ml de meio de NIH 3T3 (DMEM + 10% FBS). </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Ressuspender as células com 10 ml de NIH 3T3 médio e transferir para um 10 centímetros prato de cultura. </li><li> Incubar as células em uma temperatura de 37 ° C, 5% de CO2. É muito importante que as células NIH 3T3 são sempre mantidas underconfluent (50-60%), tal como a frequência de transformação espontânea (e, por conseguinte, níveis basais de formação de focos) aumenta uma vez que uma cultura atinge a confluência. </li><li> Semente de 3 x 10 5 células por placa de 10 cm e incubar O / N para a infecção, no dia seguinte. </li></ol></li><li> Infecção <ol><li> Colher o sobrenadante retroviral do prato Plat-E usando uma seringa descartável de 10 ml, filtração através de um filtro de membrana de nylon de 0,45 um de tamanho de poro, e transferindo-o para um 15 mltubo. </li><li> Adicionar 10 ml de meio Plat-E fresco às células (sem antibióticos), e devolvê-las para a incubadora. </li><li> Aspirar o meio do prato de células NIH 3T3 de estarem infectados, e adicione 5 ml de meio de NIH 3T3 regular e 5 ml de sobrenadante filtrado que contém o vírus. </li><li> Adicionar ao prato polibreno a uma concentração de 6 ug / ml. </li><li> Incubar as células S / N a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, repita os passos 3.2.1 - 3.2.5 para uma segunda rodada de infecção. </li><li> Substituir o meio contendo vírus NIH 3T3 com meio regular. </li><li> Permitir que as células NIH-3T3 para crescer durante 2-3 semanas, substituindo o meio, conforme necessário. Verifique a expressão da proteína de interesse por electroforese e imunotransferência de proteína convencional em lisados ​​de células inteiras a partir de placas réplica. </li></ol></li></ol> 4. Coloração e Quantificação <ol><li> Violeta Cristal Coloração <ol><li> Aspirar o meio de NIH 3T3 e colocar os pratosem gelo. </li><li> Lavam-se as placas duas vezes com PBS gelado. </li><li> Fixar as células com metanol gelado durante 10 min. </li><li> Remover os pratos de gelo, aspirar o metanol, e adicionar 3 ml de solução de violeta cristal 0,5% feitos em 25% de metanol (RT). </li><li> Incubar as placas durante 5 min à TA. </li><li> Aspirar a solução de cristal violeta e lavar cuidadosamente o prato com Milli-Q H2O até sem cor sai na lavagem. </li><li> Permitir que os pratos para secar O / N em uma bancada (descoberto). </li></ol></li><li> Focos Quantificação <ol><li> Uma vez que os pratos estão secos, use uma régua para medir as coleções de cor escura de células na placa. Contar apenas aqueles que são maiores do que 5 mm de diâmetro, como &quot;focos&quot;. Anote o número de focos e calcular médias e a importância em comparação com os controles. </li></ol></li></ol>

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A FFA fornece um método rápido e fácil de avaliar a transformação maligna in vitro. É passível de exibição de um número relativamente grande de genes candidatos e seus requisitos técnicos modestos torná-lo rentável. Além disso, dois ou mais genes pode ser co-expressa (por vezes referido como um ensaio de &quot;cooperação&quot;) para avaliar o potencial tumorigénico de combinação. As vantagens deste ensaio contar com a sua técnica simples, a sua facilidade de quantificação e a sua relativam...

As células tumorais podem ser distinguidos dos seus homólogos normais por uma ampla gama de alterações, a partir de padrões de expressão de genes para epigenomics a alterações morfológicas e proliferativas. Entre estes últimos, redução da dependência do soro, a perda de contacto (densidade) de inibição, a aquisição de proliferação independente de ancoragem e, em última análise, a capacidade de formar tumores quando injectadas em animais são indicadores úteis, mensuráveis ​​de transformação maligna 3. Vários in vitro e em ensaios in vivo têm sido desenvolvidos para a transformação celular. Os ensaios in vitro por objectivo a identificação e medindo alterações na morfologia da cultura (ensaio de formação de foco), dinâmica de cultura (taxa de crescimento, a densidade de saturação) e o factor de crescimento (crescimento em soro reduzido) ou ancoradouro (crescimento em agar mole) requisitos. O padrão de ouro para a determinação do carácter maligno de um tipo de célula permanece a formação de tumores (xenoenxertos) em animais experimentais. No entanto, tele custa e comprimento de estudos in vivo nem sempre fazê-los justificável como uma primeira etapa de validação ou screening de oncogenes candidatos. Embora nenhum ensaio in vitro fornece uma avaliação definitiva do potencial oncogénico de um gene, que não fornecem informações sobre potencial oncogénico que pode afinar futuro em estudos in vivo. Um dos sistemas mais utilizados para a avaliação do potencial oncogênico in vitro é o foco Formação Ensaio 2. Esta abordagem baseia-se na utilização de NIH 3T3 de fibroblastos de rato, uma linha de células não transformadas que mostram forte inibição de contacto. A sobre-expressão de um oncogene resulta em perda do crescimento dependente de densidade; As células transformadas podem então crescer em múltiplas camadas, formando &quot;focos&quot;, facilmente visualizado contra a monocamada de células não transformadas fundo. O Ensaio de formação de focos, em seguida, mede a capacidade de um oncogene candidato para induzir a transformação maligna, como evidenciado pela perda de contacto Inibiç como um fenótipo mensurável. O FFA tem sido utilizado para avaliar a sobre-expressão por transformação de proteína-quinases (por exemplo, Src 4, 5 BRAF), factores de transcrição (por exemplo, N-myc 6) receptores, L-acoplados à proteína (por exemplo, P2RY8 7) e GTPases (p.ex. , Ras 1), entre outros. A relativa facilidade deste ensaio faz-se uma boa escolha que vai proporcionar uma resposta rápida e visualmente claro para se sobre-expressão do gene é suficiente para transformar células de fibroblastos NIH 3T3 de rato in vitro. O FFA descrito neste protocolo utiliza a linha celular de empacotamento Plat-E 8, o qual fornece as proteínas virais de empacotamento, e o vector retroviral pBABEpuro 9 (Addgene plasmídeo 1764) para a produção de retrovírus. Após transfecção com a construção pBABEpuro contendo o gene de interesse, a linha celular Plat-E vai produzir retrovírus ecotrópico que pode ser utilizado para infectar as células NIH 3T3. Tseu método de entrega gene viral é mais eficiente do que os métodos de transfecção química tradicionais e oferece uma maneira de expressar de forma sustentável o gene 10. Uma vez incorporado no genoma das células NIH 3T3, a sobre-expressão do gene de interesse é impulsionado pelo repetições terminais longas virais (LTR) 11. Esta expressão constante pode ser usado para determinar se o gene de interesse tem actividade oncogénica, tal como medido pela formação de focos, sobre as células NIH 3T3.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
		</tr>
	</tbody>
</table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 é um fator fundamental zipper hélice-alça-hélice leucina (bHLHZ) de transcrição que é um membro da MYC / MAX / network MAD. Ele é um membro da família atípica MAD 13-15, e tem sido referida como estando envolvida na carcinogénese 16,17. Quando comparado com pBABEpuro (controlo negativo) e MYC (controlo positivo), os pratos de NIH 3T3 que sobre-expressas foi MXD3 tinham significativamente menos focos (Figura 1A). Os dados na Figura 1B foram recolhido...

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Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

O ensaio de formao de foco fornece um método simples para avaliar o potencial de transformação de um oncogene candidato.

Concentre Formação: Um Ensaio Celular para Determinar o Potencial oncogénica de um Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

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27.7K visualizações.  University of California, Davis.  O ensaio de formao de foco fornece um método simples para avaliar o potencial de transformação de um oncogene candidato. 

Vídeo: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarcadores em um modelo animal para Revelando correlatos neurais, hematológica e Comportamental do TEPT

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicina

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

High-throughput Citometria de Fluxo Ensaio Celular para detectar anticorpos em N-metil-D-aspartato ou dopamina-2 Receptor em Soro Humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

A modificação<em&gt; In vitro</em&gt; Invasion Ensaio para determinar o possível papel dos hormônios, citocinas e / ou fatores de crescimento na Cancer Mediating Invasion celular

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

A Novel<em&gt; In vivo</em&gt; Técnica Gene Transferência e<em&gt; In vitro</em&gt; Ensaios baseados em células para o Estudo da perda óssea em Distúrbios Osteomusculares

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensional (3D) Tumor Esferóide Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilização do ensaio de formação de colónia agar mole para identificar inibidores de tumorigenicidade em células de cancro da mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

VERDADEIRO silenciamento gênico: Triagem de um heptâmero tipo Guia de pequeno RNA Biblioteca de agentes terapêuticos potenciais Câncer

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Injeção de células Singênico Murino melanoma para determinar o seu potencial metastático nos pulmões

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

Um modelo baseado em células epiteliais do plexo coróide da Barreira Fluid Human Blood-cefalorraquidiano para o Estudo de infecção bacteriana do lado basolateral

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...