Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

Declaração de Ética: Procedimentos envolvendo indivíduos animais foram aprovados pelo Institutional Animal Care e do Comitê Use na Stanford University School of Medicine. NOTA: Os animais estão sob anestesia com 3% de isoflurano vaporizador e fornecimento 3% de O2 gás. O uso de pomada veterinário nos olhos pode ajudar a prevenir o ressecamento enquanto sob anestesia. 1. Preparação de células <ol><li> Crescer endoteliais e células pericyte culturas humanas em placas de 100 mm com 10 ml de a mídia apropriada. Substituir 10 ml de meio fresco a cada 2 - 3 dias até que as células atingir 80% de confluência. <ol><li> Cultura de células endoteliais (ECs) em meio completo de células endoteliais (ECM) suplementado com empresa fornecido 5% de soro fetal de bovino (FBS), 10% de penicilina / estreptomicina e suplemento de crescimento celular endotelial 10%. </li><li> pericitos cultura em meios pericyte completo (PM) suplementado com empresa forneceu 2% FBS, 10% de penicilina / estreptomicina e suplemento de crescimento pericyte 10%. </li&gt; </ol></li><li> Preparar misturas de células <ol><li> Calcula-se a quantidade necessária de células para os grupos experimentais e de controlo. NOTA: Cada plug no grupo experimental contém um milhão (1 x 10 6) ECs humana e duzentos mil (2 x 10 5) pericitos. Para o grupo controle, cada vela contém apenas 1,2 milhão de ECs humanos. O grupo de controlo negativo tem gel matriz única. Cada grupo deve incluir, pelo menos, quatro tomadas de corrente, o que exigiria dois ratos como tampões podem ser injectadas em cada lado de um rato parte inferior das costas. Três tampões serviria como triplicado para a experiência e o quarto pode ser utilizado no caso de uma falha. Preparar 25% de células adicionais para coincidir com o volume adicional de gel. </li><li> Recolher e contar as células de placas de cultura. <ol><li> Para retirar as células, remova a mídia e lavar uma vez com fosfato de 1x temperatura ambiente salino (PBS). Remover PBS, adicionar 1 ml de 0,25% EDTA de tripsina / 2,21 mM, e incubar durante 1 min. Lave as células fora da placa por adição de 2ml de meio e recolher num tubo de 15 ml. </li><li> A contagem de células utilizando um hemocitómetro de acordo com as instruções do fabricante. </li><li> Centrifuga-se o número adequado de células numa pelete a 400 xg durante 5 min. Para o grupo experimental, de centrifugação tanto ECs e pericitos para baixo junto em um pellet. </li></ol></li></ol></li></ol> 2. Preparação matriz de gel <ol><li> Descongelar completamente na matriz de gel a 4 ° C durante 4 horas ou durante a noite para se obter uma solução homogénea. Não é necessária mistura. </li><li> aliquota gel de matriz em tubos de 1,5 ml e armazenar a 4 ° C. </li><li> Pré-frio estéreis tubos de 1,5 ml e 28 g seringas de 1 cc de insulina, a 4 ° C. Cuidado: Mantenha gel matriz no gelo em todos os momentos. </li><li> Misturar na matriz de gel frio com bFGF a uma proporção em volume de 1: 100 (concentração final de bFGF = 0,5 ug / ml; bFGF solução estoque = 50 ug / ml) após reconstituição solução de gel matriz por pipetagem para cima e para baixo várias vezes, incubar em gelo por 30 - 60 min bntes de mistura com as células no passo 3.1. NOTA: Descongelar o estoque bFGF antes da mistura. Calcular o número de fichas necessárias para a injecção. Por exemplo, cada plug requer 200 gel matriz ul. Por conseguinte, preparar 25% de gel adicional. </li></ol> 3. Misturar matriz de gel com células <ol><li> Para cada grupo, ressuspender o sedimento de células (número de células contendo o caso) com o volume calculado de gel de matriz contendo bFGF. Misture com cuidado para evitar a formação de espuma e deixe no tubo de 15 ml em gelo até ratos são preparadas para injecção. </li></ol> 4. Rato Preparação <ol><li> 1 Anestesiar ratos SCID durante 5 minutos em 3% de isoflurano mais de O 2 a 3% em uma câmara de indução. Concentre-se em um grupo experimental, ratos ou 2, de cada vez. </li><li> Remover o rato da câmara de indução, lugar em um lado ventral almofada de aquecimento para baixo e rapidamente anexar um bocal focinho até o rosto do rato através de um sistema não-reinalação para fornecer anestesia em todo o injeprocedimento cção. Comutar o fluxo de gás a partir da câmara de indução para o sistema de reciclagem do ar e não diminuir a pressão do gás de 1,5% de isoflurano e 1,5% de O 2. </li><li> Remover pêlos de ambas as regiões traseiras laterais (cerca de 1 cm de diâmetro) com um creme da remoção da máquina de barbear ou cabelo. </li><li> Limpar a área da pele com uma compressa de álcool de 70%. </li><li> Devolver o mouse para a câmara de indução e repita os passos 4,2-4,4 com o segundo mouse. </li></ol> 5. Injecção matriz de gel <ol><li> Prepare um rato para injecção, anexando ao sistema não-reinalação e colocando no lado da almofada de aquecimento ventral para baixo. </li><li> Carregar o gel matriz para uma pré-arrefecida de 28 g de 1 cc de insulina seringa um rato de cada vez. <ol><li> Carregar o volume de 500 ul de gel de matriz completa em dois tampões (200 ul por encaixe e ainda 25% adicionais) para dentro da seringa. </li><li> Certifique-se para injectar o gel matriz para os ratos dentro de 3 min de carregar as células para dentro da seringa para impedir o cells se deposite no lado da seringa e para impedir a matriz de gel solidifique no interior da seringa. </li></ol></li><li> Levantar a pele volta para localizar espaço subcutâneo, em seguida, injectar 200 ul de gel matriz lentamente e uniformemente no espaço subcutâneo do posterior para trás da caixa torácica. </li><li> Remover a agulha de injecção lentamente, tendo o cuidado de evitar a matriz de gel de vazar para fora do local de injecção. Uma colisão vai formar no local da injecção. Limpe local da injecção com uma compressa embebida em álcool. </li><li> Repita os passos de injecção 5,2-5,5 no outro lado da parte traseira do rato, em seguida, deixar o mouse sobre as suas costas para 1 min para permitir a gelificação do gel matriz sobre a almofada de calor. </li><li> Delinear as duas colisões usando um marcador permanente para identificar a colisão após 14 dias. Depois de algum cabelo volta a crescer, será mais fácil encontrar a colisão. </li></ol> 6. Matrix Gel plug Isolamento: 14 dias após a injecção <ol><li> Eutanásia com humanidade os ratos por exposição do animals a&gt; 5 min de inalação de dióxido de carbono, seguido por deslocamento cervical para assegurar a morte. </li><li> Retire o bujão de gel matriz das costas do mouse. <ol><li> Remova cuidadosamente o cabelo de novo cultivadas em torno do local da injecção onde a ficha está localizado repetindo o passo 4.3. A linha de marcação indica o tamanho da colisão original sob a pele. </li><li> Excisar o encaixe juntamente com a pele e os músculos que rodeiam as camadas unidas nos lados superior e inferior do tampão, respectivamente. <ol><li> Usando finas tesouras cirúrgicas, fazer uma incisão na fronteira acentuada do tampão que é perpendicular à pele e cortar ao músculo debaixo do bujão. Em seguida, cuidadosamente cortada em torno da periferia do tampão ao longo da fronteira marcada. </li><li> Retirar a ficha, mantendo-ensanduichada entre as camadas de pele e músculo. lave imediatamente em um pequeno copo com 1x PBS para lavar o excesso de sangue. A ficha está agora pronto para ser fixado (passo seguinte, 6.3). </li></ol></li></ol></li><li> Corrigir os plug em 4% de paraformaldeído. <ol><li> Colocar todo o tampão, incluindo as camadas da pele e músculo, em um tubo de 50 ml com 25 ml de 4% de paraformaldeído durante 24 horas à temperatura ambiente sem agitação. No dia seguinte, transferir o plugue em 25 ml de etanol a 70% durante mais 24 horas antes da inclusão em parafina. NOTA: Punho paraformaldeído com cautela. Use luvas. </li></ol></li></ol> 7. Parafina Processo Matrix Gel plug <ol><li> Prepare o plug para inclusão em parafina. <ol><li> Orientar a ficha, como mostrado na Figura 1A; colocar a ficha numa cassete de tecido de modo que o lado da ficha é virada para cima e as duas camadas da pele e músculo são visíveis através da superfície do bloco de tecido que vai ser cortado em primeiro lugar durante o corte. </li></ol></li><li> Parafina incorporar a ficha de acordo com Parafina protocolos padrão 13. </li><li> Corte 8 - 10 mm de espessura fora do bloco de tecido e montar em lâminas de microscópio According a parafina seccionamento protocolos padrão 13. </li><li> Armazenar as lâminas em local seco e fresco à temperatura ambiente até que esteja pronto para a coloração. </li></ol> 8. Secções mancha com hematoxilina e eosina (H &amp; E) 13 <ol><li> De-paraffinize as secções de tecido por passagem das lâminas por meio de 100% de xileno, duas vezes, com 100% de etanol, 95% de etanol, 70% de etanol e, finalmente, 1 x PBS, cada um durante 5 min à temperatura ambiente. Deixar as lâminas em 1x PBS até ao passo seguinte. </li><li> solução de pipeta 100 mL H &amp; E para a seção e incubar por 1-5 min, ou até que haja um claro contraste entre núcleos azuis e citoplasma rosa em uma célula como visto sob um microscópio de luz na posição vertical no aumento de 10x. Tenha cuidado para não deixar que qualquer solução H &amp; E tocar o objectivo. </li><li> solução de lavagem H &amp; E fora do slide, molhando o slide em um grande copo de água da torneira, em seguida, coloque o slide em um rack no copo e lave com água corrente durante 2 min. </li><li> Move para o passo 9.8 para montar os slides. </li></ol> 9. Stain outros slides para Capilares Humanos e pericitos <ol><li> De-paraffinize as secções de tecido por passagem das lâminas por meio de 100% de xileno, duas vezes, com 100% de etanol, 95% de etanol, 70% de etanol e, finalmente, 1 x PBS, cada um durante 5 min à temperatura ambiente. Deixar as lâminas em 1x PBS até ao passo seguinte. </li><li> Ferva as seções de solução de recuperação antigênica. <ol><li> Em um copo de 2 L, preparar a solução de recuperação de antigénio de ebulição usando 1x tampão de citrato (pH 7,0) a 95 ° C num bloco de aquecimento. </li><li> Depois de ferver, coloque as lâminas em um rack de metal e mergulhe na solução de recuperação antigênica em ebulição durante 20 min. </li><li> Cuidadosamente remover o copo do bloco de aquecimento com as lâminas submersas e ainda permitir que a solução voltar lentamente à temperatura ambiente durante cerca de 30 min. </li><li> Colocar as lâminas em 1x PBS até o próximo passo. </li></ol></li><li> Bloquear as seções em tampão de bloqueio (1x PBS, 5% de soro de cabra, 00,3% de Triton X-100). <ol><li> Desenhar um círculo hidrófobo em torno das bordas de cada secção com uma caneta PAP. </li><li> Colocar as lâminas em uma bandeja de humidade com água destilada na parte inferior do tabuleiro. </li><li> Pipetar 100 ul de tampão de bloqueio para as secções assegurando-se todo o tecido é coberta por fluido (pode necessitar de mais de 100 uL). </li><li> Incubar secções com tampão de bloqueio durante 1 h. </li></ol></li><li> Incubar seções com anticorpo primário. <ol><li> Preparar uma solução com dois anticorpos primários para sondar cada secção para CD31 (ECS) e actina do músculo liso (pericitos) diluída em tampão de diluição (PBS 1x, 1% de BSA, 0,3% de Triton X-100). NOTA: Aqui, a concentração de anti-CD31 é de 1:50 e concentração actina anti-músculo liso é de 1: 300. </li><li> Incubam-se as secções durante a noite a 4 ° C com 100 ul de anticorpos primários em uma bandeja de humidade contendo água destilada. </li></ol></li><li> Lavam-se as lâminas três vezes em 1x PBST (PBS 1x + 0,1% TweEN20) durante 5 min cada lavagem. </li><li> Incubar seções com anticorpo secundário. <ol><li> Prepara-se uma solução com um anticorpo secundário anti-coelho de cabra conjugada com fluoróforo para reconhecer o anticorpo de coelho anti-CD31 diluído no tampão de diluição. <ol><li> NOTA: O anticorpo primário actina de músculo liso já é conjugado com um fluoróforo CY3, assim, não coloração do anticorpo secundário é necessária. coelho concentração de anticorpo secundário de cabra anti-é de 1: 250. </li></ol></li><li> Incubar as secções durante 1 hora com anticorpo secundário ul 100 na bandeja de umidade contendo água destilada. </li></ol></li><li> Lavam-se as lâminas três vezes em 1x PBST, durante 5 min cada lavagem. </li><li> Montar os slides. <ol><li> Pipetar 2 - 3 gotas (~ 40 - 60 L) de solução antifade montagem com coloração nuclear DAPI em cada secção. </li><li> Delicadamente, coloque um não. 1,5 tampa de deslizamento sobre as seções e solução de montagem, permitindo que o fluido para se espalhar sobre as seções e as bordasa lamela. Suavemente utilizar um dedo para pressionar para fora as bolhas de ar que podem ser localizados ao longo do percurso. </li><li> Deixe que as lâminas secar por pelo menos 1 hora antes de utilizar. </li></ol></li></ol> 10. Quantificar Capilar Densidade e Estrutura 14 <ol><li> Contar o número de capilares em H &amp; E secções coradas, expressa como # / mm 2. <ol><li> Adquirir fotos de quatro campos diferentes em um microscópio de luz na posição vertical sob 40X. NOTA: Os capilares são identificados como tubos cheios com células vermelhas do sangue. </li><li> Alternativamente, em vez de capilares contando manualmente, use imagem software de análise (por exemplo, Wimasis Análise de Imagem) para quantificar vários parâmetros de angiogênese, como o número de navio, comprimento médio navio e diâmetro, e pontos de ramificação. </li></ol></li><li> Analisar a estrutura capilar em seções de imunofluorescência corados. <ol><li> Adquirir fotos de quatro campos diferentes em um microscópio de fluorescência invertidacom cubo de filtro FITC e TxRed. <ol><li> Use um cubo FITC para a imagem ECs por excitação a 488 nm de comprimento de onda e usar um cubo TxRed de pericitos de imagem por excitação a 594 nm de comprimento de onda. <style type="text/css">p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Arial}
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No competing financial interests enclosed.

O ensaio de tampão de gel de matriz tem provado ser um método conveniente e poderoso para avaliar a regulação do gene na angiogénese, os compostos angiogénicos e anti-angiogénicos in vivo, e para complementar os testes in vitro. Aqui, descrevemos em detalhe um ensaio tampão de gel matriz novela de angiogênese humana que investiga a interação entre as células endoteliais e pericitos durante a formação de vasos. Existem alguns passos novos e críticos n...

A angiogénese é o processo pelo qual novos vasos sanguíneos são formados a partir de uma rede vascular pré-existente e 1 é o foco da pesquisa em curso em muitas áreas, desde o desenvolvimento normal da doença. Este processo dinâmico envolve a proliferação e migração de células endoteliais (ECS) e recrutamento de pericitos para a construção de um tubo vascular que é dirigida para o local que necessita de oxigénio e de nutrientes de entrega 2. Para estudar este processo requer um ensaio dinâmico igualmente, o mais importante que pode recapitular a natureza tridimensional de formação de tubos. Os ensaios in vitro de matrizes 3D foram desenvolvidos para atender a essa necessidade e têm funcionado bem para permitir que pesquisadores para definir os passos discretos no espaço e no tempo em que a angiogénese ocorre 3, 4, 5, 6. No entanto, estes modelos in vitro de matrizes 3D estão limitadas a estudar navios não-perfundidos umd, por conseguinte, não têm componentes críticos pertinentes para o processo de angiogese (por exemplo, circulando o crescimento e factores inibitórios, a tensão não natural / forças através do leito vascular) e falhar para simular o ambiente complexo presente no tecido vivo. Para resolver esta limitação, vários ensaios in vivo da angiogénese têm sido desenvolvidos 7, incluindo o ensaio de tampão de gel de matriz, que será o foco do nosso relatório 8, 9. O ensaio de matriz de tampão de gel é um ensaio in vivo de angiogénese bem estabelecido que atrai pesquisadores, pois proporciona uma plataforma robusta para testar os papéis dos diferentes células e substâncias na angiogênese. gel de matriz é uma membrana basal solução disponível comercialmente que é segregado pela linha celular de Engelbreth-Holm-Swarm (EHS) sarcoma de rato que solidifica em um material semelhante a gel a 37 ° C. A matriz de gel pode ser misturado com as células e / ou substâncias, tais como factores de crescimento, e injected subcutaneamente no mouse. O anfitrião ECs vai invadir a ficha ao longo de 14 dias, formar uma rede vascular, e tornar-se perfusão com sangue do hospedeiro. Até à data, os ensaios camada de gel de matriz têm-se centrado exclusivamente no estudo do comportamento da célula endotelial durante a angiogénese, no entanto, com o melhor de nosso conhecimento nenhum esforço foi feito ainda para determinar se este ensaio pode ser utilizado células endoteliais para co-cultura e pericitos para estudar como estes dois tipos de células interagem durante a angiogênese. Especificamente, a compreensão da relação entre ECs e pericitos é valioso para o estudo de doenças em que a perda de vasos sanguíneos é patológico, incluindo isquemia microvascular e doença vascular periférica 10, 11, 12. Aqui, nós descrevemos um protocolo que introduz pericitos derivadas de humanos para a mistura de gel da matriz juntamente com ECs humano e factor de crescimento de fibroblastos (bFGF). Esta mistura pode então ser injectado por via subcutânea in o dorso de ratinhos SCID para permitir a formação de pericitos, revestidos, vasos híbridos totalmente funcionais. Nosso protocolo descreve como preparar tampões de gel matriz contendo ECs humanos com ou sem pericitos humanos, colocação em ratinhos SCID e como analisar os cortes histológicos para endpoints angiogênese críticos.

<table border="0" cellpadding="0" cellspacing="0" width="806">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Representante H &amp; E e imunofluorescência coloração das seções camada de gel de matriz são mostrados na Figura 2. Os pontos de CE apenas fichas de exibir alguns vasos que na maioria não são perfundidos com sangue (Figura 2 parte superior esquerda, setas pretas), enquanto fichas contendo tanto ECs e pericitos mostrar vários perfundido vasos com diâmetros maiores e cobertura pericyte completa, como evidenciado por coloração SMA positivo imed...

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Nós apresentamos um protocolo para estudar as interações endotelial-pericyte humanos no rato usando uma variação do ensaio plugue angiogênese gel matriz.

No estudo in vivo da Human Interaction Endotelial-pericyte usando o plug Ensaio Matrix Gel no mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

in-vivo-study-human-endothelial-pericyte-interaction-using-matrix-gel

Research

JoVE Journal

Biology

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

9.6K visualizações.  Stanford University.  Nós apresentamos um protocolo para estudar as interações endotelial-pericyte humanos no rato usando uma variação do ensaio plugue angiogênese gel matriz. 

Vídeo: In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Teste de sensibilidade visual para a velocidade e direção do movimento em Lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biologia

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Otimizado de fibrina Gel Assay Bead para o Estudo da angiogênese

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

In vivo e in vitro de Estudos Adaptor-clathrin Interação

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

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. ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself Dispositivo de Recuperação de amostras criopreservadas cair acidentalmente em Tanques de armazenamento criogênico

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modelagem do Morbid Genoma Humano utilizando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

De alta resolução de imagem Time-lapse e análise automatizada de microtúbulos Dynamics em Viver umbilical humano Células Endoteliais da Veia

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap juncional intercelular comunicação: um biomarcador funcional para avaliar os efeitos adversos de tóxicos e toxinas, e Benefícios para a saúde de produtos naturais

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...