Apoiado em parte por uma concessão do National Cancer Institute 1R03CA172923.

Declaração de Ética: O uso de camundongos em pesquisa só é aceitável em casos em que pode avançar a compreensão humana da biologia fundamental ou para a eventual tratamento da doença. 1. Ordenação <ol><li> Compra fêmea C57BL / 6 murganhos (idade de 6 - 8 semanas), 5 ratinhos por grupo. Permitir vários dias para os ratos para se ajustar. </li></ol> 2. Preparação de células B16-BL6 <ol><li> Remover placas de cultura de células de incubadora a 37 graus e coloque em uma capa estéril. As placas devem ser de aproximadamente 70% confluente com células de melanoma murino B16-BL6. Uma maior confluência as células podem alterar o potencial metastático. </li><li> Adicionar 1 ml de 0,05% de tripsina-EDTA por placa, deixar repousar durante 1 min, e depois aspirar a solução de tripsina-media. </li><li> Adicionar 1 ml de tripsina por placa e retornar as placas ao incubador durante 10 min. </li><li> Depois de mover as placas da incubadora para a capa estéril, adicionar 4 ml de livre de soro Rosewell Park Memorial Institute Medium (RPMI) para cada placa. </li><li> Coletar as células com uma pipeta estéril, motorizada. Ejectar as células contra a parte inferior da placa, com a ponta pressionado em um muito ligeiro ângulo, para romper as células. Repetir várias vezes a fim de assegurar uma separação adequada de aglomerados de células, o que pode de outro modo entupir a agulha de ejecção. Lembrar e transferir para um tubo de 50 ml. </li><li> Usar um hemocitómetro para determinar a densidade celular. Mantendo o número total de células B16-BL6 constante, determinar o volume de meio RPMI isento de soro necessária para atingir concentrações finais de 0, 0,065, 0,015, 0,25, e 0,5 milhões de células / ml. </li><li> Igualmente Alíquota da mídia no tubo de 50 ml a 15 ml tubos cônicos. Centrifugar os tubos cónicos de 2250 xg durante 5 min. Decantar a mídia. Adicionar um volume apropriado de meio RPMI isento de soro e confirmar a densidade de células desejada através hemacitómetro. Ajustar densities- adicionando RPMI adicional ou fiação e ressuspensão num volume- menor em conformidade. </li><li> Alíquota de 500 ulde cada suspensão de células marcadas em 1,8 ml de tubos em gelo. </li></ol> 3. Cauda injecção na veia <ol><li> Tome um rato, realizada pela cauda, ​​e guiá-lo traseira pela primeira vez no restrainer mouse. Com o tronco na câmara principal do limitador, com a cauda do lado de fora, retirar o rato para o fim do limitador. Insira o restrainer-êmbolo, continuando a empurrar até que o mouse é segura. </li><li> Rodar o rato 90 graus de modo a que uma das veias lateral da cauda esteja voltada para cima. Usando uma compressa embebida em álcool, limpar vigorosamente o lado da cauda do rato, em particular quando a veia da cauda é mais visível. Nota: Uma boa iluminação é essencial para visualização veia da cauda eficaz sobre B57BL / 6J. Lâmpadas de calor ou almofadas cirurgia aquecida, embora não necessário, também vai ajudar a aumentar o volume da veia da cauda. </li><li> Num capuz de cultura estéril, recolher 300 uL de meio de cultura celular a partir do tubo de 2 milhões de células / ml. Inverta a agulha, sacudindo as sIDE e empurrando o êmbolo, para ejectar bolhas a partir da seringa. Ejectar cultura RPMI-a resultar num volume final de 250 ul. </li><li> Estender o rato-cauda com a mão não-dominante. Segurar a cauda com a extremidade proximal da cauda elevada com o dedo indicador da mão não-dominante, e a parte distai da cauda deprimido com o polegar. Nota: Neste modo, a veia da cauda é mantido numa posição lateral, paralela à superfície do limitador, com uma entrada congruente possível na parte mais distante da cauda. </li><li> Inserir a agulha na veia da cauda, ​​no sentido da extremidade distai, com um ângulo descendente minutos para começar, e, em seguida, ajustar a agulha para corresponder ao ângulo da veia da cauda, ​​após nova inserção (baixando extremidade do êmbolo do parente de uma seringa à agulha) . </li><li> Inserir a agulha de aproximadamente 1 cm na veia da cauda. Após a inserção, começam a empurrar o êmbolo, que prende a agulha constante. Nota: Se a agulha se encontra na veia da cauda e a extremidade da agulha é not obstruído os meios de comunicação deve fluir sem obstáculos na veia. injecção eficaz é caracterizada por depuração do sangue a partir da veia. Se houver resistência, ou uma bolha começa a formar no local da injecção em, remover a agulha e tentar de novo numa localização mais proximal ao longo da veia da cauda. </li><li> Retire a agulha da veia e descartá-lo. Segure gaze contra o local de entrada para evitar qualquer sangramento. </li><li> Retirar o êmbolo a partir do limitador e colocar o rato na gaiola destinatário. </li><li> Repita o procedimento para todas as outras concentrações. Nota:. Lung estabelecimento focos demora cerca de 2-3 semanas, dependendo da variabilidade da linha celular e a dimensão desejada dos focos 9 No entretanto, os murganhos devem ser monitorizados quanto a sintomas que foram sacrificados no início, como ânsia excessiva. </li></ol> 4. Contando o Lung Focos <ol><li> Euthanize o mouse, colocando em uma câmara de CO 2 e expondo a 100% CO 2 </sUB&gt; introduzido a 10-30% do volume da câmara por minuto durante 5 minutos. Siga com uma luxação cervical. </li><li> Afunilar o mouse em isopor. Usando uma tesoura cirúrgica, criar uma incisão cirúrgica ao longo do esterno. </li><li> Fazer duas incisões adicionais, tanto da parte superior do esterno e incisão de ramificação para os ombros do rato, expondo a caixa torácica. </li><li> Fazer dois cortes, cada um ao longo dos lados laterais do esterno, para remover a caixa torácica a partir do tronco, expondo os pulmões e coração. </li><li> Segurando o coração com pinças cirúrgicas, corte o esôfago e traquéia, liberando o coração e os pulmões do mouse. Colocar sob um microscópio de dissecação, a 10X de ampliação, para melhor visualização. </li><li> Dissecar o coração dos pulmões e remover o timo, deixando apenas os dois pulmões. </li><li> Com os pulmões sob um escopo de dissecação, começa a contar o focos de pulmão. Cada focos deve aparecer como uma obstrução ao fluxo circular, Brown na superfície dos pulmões. <ol><li> Paraconsistência entre ratos, contar o número de focos na superfície de um lobo, e depois inverta que lóbulo. Fazendo cada um dos quatro lobos permite individualmente para contagem mais fácil. </li></ol></li><li> Salvar e corrigir os pulmões se realizar IHC, caso contrário, descarte os restos. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Circulating células tumorais a partir de uma injecção na veia da cauda representam as metástases que escapam locais de crescimento primário e migrar através de vias como a corrente sanguínea ou no sistema linfático, ocasionalmente, estabelecendo-se nos leitos capilares de locais distantes. 14 O protocolo descrito acima serve como um modelo para secundário metástases. Com um número óptimo de células B16-BL6 injectadas, experimentadores pode determinar o efeito relativo de um fármaco administrado ou população de células imune, pós-neutralização, sobre a metástase de células de cancro. Este protocolo tem limitações. As células tumorais em circulação foram seleccionados pela sua capacidade para metastizar e são, por conseguinte, não imunogénico, tendo baixos níveis de histocompatibilidade principal de classe 1 do complexo moléculas. 12 Além disso, a introdução súbita e agrupado de grande número de metástases é ao contrário do cenário típico em pacientes em que um pequeno número de células tumorais metastáticasdissipam ao longo de um período de tempo prolongado a partir do local do tumor primário. Além disso, as células tumorais são introduzidos por via intravenosa, a partir de cultura de tecidos e não de uma origem do tumor primário. Por isso, estas células são ao contrário das metástases secundárias, que resultam de alterações na adesão celular, a migração, o reconhecimento imunológico e estabelecimento órgão destinatário 15,16. Um protocolo alternativo seria a injecção subcutânea de células B16 / BL6 para a geração de tumores primários e a análise dos resultantes metástases pulmonares &quot;espontâneas&quot;. Essas metástases &quot;espontâneos&quot; foram encontrados para conter mais heterogeneidade do que suas contrapartes &quot;experimentais&quot;, combinando mais de perto aqueles de pacientes humanos. O protocolo está a limitar na sua capacidade para determinar a influência de um composto experimental em metástase. Com uma injecção na veia lateral da cauda, ​​o número de células de melanoma injectado na corrente sanguínea pode ser aproximada através hemocytometer. Com metástase &quot;espontânea&quot;, no entanto, existe uma maior heterogeneidade entre tumores, adicionando uma variável adicional para o número de focos resultante pulmão. 16 Em conclusão, o modelo de melanoma murino B16-BL6 veia da cauda representa um modelo simples para determinar a influência de um tratamento de imunoterapia ou de metástase. Por via intravenosa injecção de células tumorais em circulação, os investigadores podem replicar, em certa medida, os pacientes pós-metástase. Considerando os efeitos destrutivos de células tumorais metastáticas a os doentes com cancro, este modelo é uma ferramenta poderosa para a compreensão do processo de passos múltiplos de metástases.

A metástase é uma das principais causas de morte entre os pacientes com doenças malignas. O processo de disseminação metastática de células cancerosas é mal compreendida, mas parece envolver vários passos, incluindo a invasão de tecidos adjacentes, intravasamento no sistema linfático e na vasculatura, a sobrevivência e a translocação dentro do sistema circulatório, o extravasamento a partir da vasculatura no local de metástases, adaptação para o novo microambiente do novo local, e colonização no novo local por proliferação e formação de tumores secundários 1. Cada um destes eventos biológicos envolvem a transformação, a difusão e a sobrevivência de células tumorais metastáticas. Por exemplo, a transformação do fenótipo epitelial da célula tumoral para um fenótipo mesenquimal, juntamente com a interacção bem sucedida com a matriz extracelular, permitir-lhes invadir tecidos adjacentes e metastizar para outras partes do corpo. 2,3 Além disso, estes metastático célulasdeve sobreviver dentro da corrente sanguínea circulante e escapar à vigilância imunitária do hospedeiro. 4,5 Finalmente, quando implantados num local distante dentro do corpo, as células tumorais têm de se adaptar ao seu microambiente, a fim de proliferar e formar tumores secundários. 6,7 Portanto , embora a metástase é um fenômeno comum entre pacientes com câncer, as células tumorais metastáticas ter várias áreas de vulnerabilidade que são passíveis de intervenção terapêutica. Dada a natureza imunogênica de melanoma, eo recente interesse em imunoterapias, os modelos de melanoma são cada vez mais útil. 8 Nos Estados Unidos sozinho, o melanoma é a causa de um número estimado de 9.000 mortes por ano. 9 locais mais comuns de metástase de melanoma são ossos, cérebro , fígado e pulmões. A maioria das metástases para locais distantes ocorrer através da corrente sanguínea. Circulating células tumorais no sangue deve escapar depuração imunológica, chegar a um leito capilar de um órgão distai einvadem através das células endoteliais do vaso sanguíneo, de modo a estabelecer-se com sucesso. 4-7,10, 11 Para imitar os fenómenos comuns e virulentas de metástase, a linha celular B16-BL6 murino foi criado. Um falecido da linha parental C57BL / 6 de células de melanoma B16-F0, B16-BL6 é o produto final de 10 sucessivas selecções para a metástase do pulmão a partir da injecção intravenosa (resultando em B16-F10), seguido por 6 selecções sucessivas para a penetração da membrana da bexiga. 12 Como tal, é uma linha celular de melanoma de confiança para a criação de focos metastáticos no rato, particularmente quando injectado por via intravenosa. 13 Após a injecção de uma quantidade suficiente de B16-BL6 células na veia da cauda de um rato B57BL 6 /, seguido por duas ou mais semanas para permitir a implantação e o crescimento das células B16 / BL6, focos metastáticos formará nos pulmões. Após a eutanásia e inspeção por microscopia dissecando, o número deindivíduo focos presente pode ser quantificada. Este, por sua vez, pode ser utilizado para estabelecer um efeito de dose-metástase, como o número de células B16-BL6 injectadas correlaciona-se com o número de focos formados na superfície dos pulmões. Este modelo é conhecido como &quot;metástase experimental&quot;, onde as células conhecidas-metastático são introduzidos directamente na corrente sanguínea, o que facilita uma disseminação e estabelecimento rápida e previsível nos pulmões, no fígado, ou outro órgão de investigação. Isto está em contraste com a &quot;metástase espontânea&quot;, onde as células tumorais são implantados, muitas vezes por via subcutânea, e metástases originam organicamente produzir células tumorais 14,15. É importante para injectar uma quantidade apropriada de células B16 / BL6 nas veias da cauda dos ratinhos. Muitos, e os pulmões será coberto com metástases e focos vizinhos, serão indistinguíveis uns dos outros. Muito poucos, e a influência de um agente terapêutico será imperceptível devido à vari inerenteções entre as injecções. A fim de obter um número óptimo de focos nos pulmões dos ratinhos C57BL / 6, que é necessário para correlacionar o número de células injectadas com a quantidade de focos metastáticos estabelecida na superfície dos pulmões, tendo em conta o distinguishability do indivíduo focos. Este protocolo irá demonstrar um modelo de melanoma metastático de murino para murganhos C57BL / 6.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Trypsin</td>
			<td style="width:275px;">Corning</td>
			<td style="width:288px;">MT25052CV</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT15040CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase Contraast Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine IV Insulin Syringes</td>
			<td>BD</td>
			<td>14-829-1D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Restrainer</td>
			<td>Braintree Scientific</td>
			<td>NC9999969</td>
			<td>Restrainer choice depends on age/size of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating Pad</td>
			<td>Harry Schein</td>
			<td>NC0012697</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

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.

Após a inspecção visual, a variação nos focos superfície é evidente. Contar o número de tumores sob um microscópio de dissecação deve produzir uma relação linear entre o número de células tumorais injectadas e o número de focos e resultando contáveis. A utilização de um número óptimo de células injectáveis, o objectivo é aquele em que os pulmões não são completamente coberto, uma vez que são em 500.000 células / ml na Figura 1A, e não muito escassamente coberto, uma vez que são em 62.500 células / ml e 125.000 células / ml. Isto também é quantificável por correlação linear (Figura 1B). <img alt="figura 1" src="/files/ftp_upload/54039/54039fig1.jpg" /> Figura 1. Lung Focos Resultante da veia da cauda injetaram células B16 / BL6. (A) Pulmão focos pode ser visualizado na superfície dos pulmões de ratinhos C57BL / 6 como massas circulares castanhos (focos representativa sob setas). Como as covasdade da cultura de células injetadas (acima de cada imagem) aumenta, o mesmo acontece com o número correspondente de resultante focos de pulmão (abaixo de cada imagem). Os pulmões do rato com a maior densidade de células injetadas tem a maior cobertura visual de focos de pulmão. As barras de escala representam 20 mm. (B) enumeração dos focos de pulmão em relação à densidade da cultura celular. Existe uma relação aproximadamente linear acima de 125.000 células / ml, conforme determinado pelo hemocitômetro. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

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

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 ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Nanyang Technological University

Research

JoVE Journal

Medicine

17.5K visualizações.  Beth Israel Deaconess Medical Center & Harvard Medical School. 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.

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Um In Vitro Sistema para Estudo de dormência Tumor ea Mude para o crescimento metastático

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Medicina

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

A Medição e Tratamento de Supressão de ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando Quantitative PCR em tempo real para determinar enxerto célula dadora num modelo murino de osso Competitiva Marrow Transplantation

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Infarto do Miocárdio e Avaliação de Resultados Funcional em porcos

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

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 ...

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.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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 ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medição da curva pressão-volume no mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Estudar o cancro do pâncreas Stem características celulares para desenvolver estratégias Novo tratamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;Patient-Like&quot; Orthotopic Singênico Rato Modelo de carcinoma hepatocelular Metástase

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Isolamento de células endoteliais progenitoras de voluntários saudáveis ​​e seu potencial migratório influenciada por amostras de soro após cirurgia cardíaca

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

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

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