Esta pesquisa foi apoiada em parte pelo programa do Instituto Nacional do Câncer dos Institutos Nacionais de Saúde Research intramuros, e pelo NCI conceder UA5CA152907.

1 Coleção de meios condicionados teste para potencial angiogênico <ol><li> Cultivar células primárias ou imortalizadas para ser testado para potencial angiogênico ou anti-angiogênico em meio de soro nativas ou baixas e recolher meios condicionados. <ol><li> Em alternativa, a utilização de meios condicionados imediatamente, ou divididos em alíquotas e armazenado a -80 ° C durante vários meses. Usar meios de soro nativo ou nonconditioned baixo como um controlo negativo, e utilizar meios não-condicionado completo de crescimento (10% FBS, ou concentração apropriada) como um controlo positivo. Como alternativa, para testar potenciais estimuladores da angiogênese, suplemento de carvão mídia despojado sem fatores de crescimento, com concentrações conhecidas de proteínas específicas de interesse e comparar com fator de crescimento mídia livre sozinho. </li></ol></li></ol> 2 Preparação de células endoteliais antes do ensaio <ol><li> Dois dias antes do ensaio, a passagem 3B-11 células para um novo frasco T-75. Número de passagem das células é importante. Este ensaio funciona melhor quando as células estão entre as segunda e sexta passagens. O ensaio não vai funcionar bem se as células endoteliais são utilizados antes da segunda ou após a décima passagem. </li><li> Crescer as células durante 24 horas em DMEM suplementado com 10% de FBS, 2 mM de L-glutamina, piruvato de sódio 1 mM, 100 U / ml de penicilina, e 100 ug / ml de estreptomicina. </li><li> Um dia antes do ensaio, soro fome 3B-11 células, como se segue: <ol><li> Aspirar mídia das células 11 3B-. </li><li> Adicionar meio de soro reduzido de DMEM suplementado com 0,2% de FBS, 2 mM de L-glutamina, piruvato de sódio 1 mM, 100 U / ml de penicilina, e 100 ug / ml de estreptomicina. </li><li> Crescer as células durante mais 24 horas adicionais. </li></ol></li></ol> 3: Preparação de reagentes antes do ensaio NOTA: As concentrações de minorias étnicas são altamente variável dependente muito. O BME não funcionam bem, se a concentração é inferior a 10 mg / ml, por conseguinte, o fabricante deve ser contactado BME antes da compra para asseguraraquisição de um lote de concentrado de forma adequada. <ol><li> Degelo reduzido fator de crescimento BME na geladeira. BME solidifica quando aquecido, por isso deve ser mantido frio antes de usar. BME pode ser armazenado sob refrigeração durante algumas semanas, ou divididos em alíquotas e congeladas a -80 ° C. </li><li> Um fator de crescimento reduzido BME é preferível a BME tradicional. A falta de fatores angiogênicos na BME irá torná-lo mais fácil de observar o efeito que os fatores do meio condicionado têm na formação do tubo. </li><li> Meios de descongelação para ser testado. </li></ol> 4 Preparação de Células Endoteliais Imediatamente antes do ensaio <ol><li> Lavar 3B-11 em células de DPBS. <ol><li> Se a visualização em microscopia confocal de fluorescência ou é desejado, antes de lavá-3B 11 células, adicionar calceina AM às células endoteliais em meio a uma concentração final de 2 ug / ml. Colocar as células marcadas calceína no escuro a 37 ° C e 5% de CO 2 antes do início do ensaio. Dissolver estoque calceï¿½aAM em DMSO e uma vez que a calceina é diluída em meio de concentração final de DMSO não deve exceder 0,1%. Após 30-45 min lavagem calceï¿½a AM a partir das células (passo 4.1) e continuar com o experimento segue. </li></ol></li><li> Tripsinizar as células por adição de 1 ml de tripsina-EDTA para frascos T-75. </li><li> Após tripsinização é completa, neutralizar a tripsina por ressuspensão células para um volume final de 10 ml em DMEM, 10% FBS, 2 mM de L-glutamina, piruvato de sódio 1 mM, 100 U / ml de penicilina, e 100 ug / ml de estreptomicina. </li><li> Células filtro através do 100 um filtro de células para remover pedaços. </li><li> Contar as células e ressuspender as células a uma concentração final de 7,5 x 10 6 células / ml em DMEM, 10% FBS, 2 mM de L-glutamina, piruvato de sódio 1 mM, 100 U / ml de penicilina, e 100 ug / ml de estreptomicina. </li></ol> Formação 5 Tubo de Ensaio <ol><li> Colocar 24 poços de placas e 1 ml de pontas em frigorífico ou em gelo durante 20-30 minutos antes de carregar a placa de modo que a BME não prematurely solidificar. </li><li> Pipetar 250 l de factor de crescimento reduzida de BME em cada poço de uma placa de 24 poços. Evite criar bolhas por não pressionar a pipeta para sua última parada, enquanto a distribuição. Tome cuidado para que suficiente BME continua no centro do poço quando as formas de menisco; Se a matriz é muito fino, apenas as células formarão uma monocamada. </li><li> Incubar placa de 24 poços a 37 ° C e 5% de CO 2 durante 30 minutos para solidificar BME. </li><li> Uma vez que a BME definiu, misturar cerca de 300 ul de meio condicionado com 10 ul de ressuspensos 3B-11 células (aproximadamente 75.000 células). <ol><li> Ajustar o volume de meio condicionado, o número de células plaqueadas dependendo do tipo de célula endotelial utilizado, e testar as diluições em série dos meios condicionados para demonstrar a actividade. Se comparando células cultivadas sob diferentes condições, normalizar condicionado volume de mídia para o número de células que foram utilizadas para condicionar os meios de comunicação. </li></ol></li><li> Tubos will começam a se formar dentro de 2-4 horas. Dependendo da concentração de factor angiogénico no meio condicionado, a formação do tubo de pico pode ocorrer entre 3 e 12 horas e o tempo do ensaio terá de ser optimizada. Se estiver a trabalhar com células endoteliais primárias, em vez de células imortalizadas, a formação do tubo inicial pode ser atrasada por várias horas. <ol><li> Tubes, muitas vezes, começam a deteriorar-se no prazo de 18 células endoteliais hr e passará por apoptose. </li></ol></li><li> No momento da formação pico tubo, mídia cuidadosamente aspirado do poço para evitar a ruptura do tubo de rede na BME. <ol><li> Lavar cada poço com 1 ml de DPBS, em seguida, aspirar. <ol><li> Para visualização imediata, adicione 1 ml DPBS frescas para cada rede tubo bem e imagem usando um microscópio invertido ligado a uma câmera digital, ou fluorescência / microscópio confocal se as células foram marcadas com calceína AM como em 4.1.1. </li><li> Para fixar a rede de tubos para posterior visualização, e adicionar paraformaldeído 4% a cadaaspirada bem e incubar durante 15 min à temperatura ambiente. <ol><li> Remover paraformaldeído, lavar duas vezes com DPBS e depois adicionar 1 mL de DPBS fresco a cada poço. </li><li> Microscopicamente imagem antes da fixação de alguns dos tubos pode quebrar durante este procedimento. </li></ol></li></ol></li></ol></li></ol> 6. Quantificação da rede de metro <ol><li> Quantificar a rede de tubos de várias maneiras diferentes, dependendo da preferência do investigador, e vários programas de computador têm a capacidade de medir os seguintes parâmetros: número de tubos; número de loops / malhas; número de locais de filiais / nós; comprimento de tubos. </li><li> Use programa de computador representante como J Imagem com o plugin angiogênese Analyzer 16 para quantificação de redes de tubos. </li></ol>

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Os autores não têm nada a revelar.

A angiogénese está envolvida em ambos os processos fisiológicos e patológicos. O estudo dos mecanismos envolvidos na angiogénese necessita da utilização de ensaios que recapitulam as etapas principais na angiogénese. O ensaio de formao de tubo endotelial oferece várias vantagens em relação a outros ensaios. É fácil de configurar, é relativamente barato, produz túbulos dentro de horas, e é quantificável. Além disso, pode ser completado em 24 ou placas de 96 poços, e, assim, pode ser utilizado para a tr...

A angiogénese, desenvolvimento de novos vasos sanguíneos a partir de vasos pré-existentes, é essencial para uma variedade de processos, incluindo o crescimento de órgãos, desenvolvimento embrionário, a cicatrização de feridas e 1-3. Recentemente desenvolvido vasos sanguíneos, revestidos por células endoteliais, suprimento de oxigênio e nutrientes para os tecidos, promover a vigilância imunológica por células hematopoiéticas e remover os resíduos 2,4. A angiogênese é de fundamental importância durante o desenvolvimento embrionário e fetal. No entanto, este processo permanece dormente no adulto, exceto durante os períodos de cicatrização de feridas, crescimento esquelético, a gravidez ou durante o ciclo menstrual 1-3. Ao longo das duas últimas décadas, os mecanismos moleculares fundamentais que regulam a angiogênese começaram a surgir. A angiogênese é um evento estritamente regulado, equilibrado pelo pró e antiangiogênicos sinais incluindo as integrinas, quimiocinas, angiopoietinas, agentes sensores de oxigênio, moléculas juncional e inibidores endógenos 5. Uma vez proangiogenic sinais tais como o factor de crescimento de fibroblastos básico (bFGF), factor de crescimento endotelial vascular (VEGF), fator de crescimento derivado de plaquetas (PDGF) e factor de crescimento epidérmico (EGF) activam os receptores de células endoteliais nas células endoteliais libertam proteases para degradar a membrana basal. As células endoteliais proliferam em seguida e migram, formando brotos a uma taxa de vários milímetros por 6,7 dias. A angiogénese está associada a várias condições patológicas, incluindo cancro, psoríase, retinopatia diabética, artrite, asma, doenças auto-imunes, doenças infecciosas, e aterosclerose 8-10. Devido à importância da angiogénese em várias doenças, compreendendo os genes e vias que regulam este processo é crítica para a concepção de melhores terapias. O ensaio de formao de tubo é um método rápido e quantitativo para a determinação de genes ou vias envolvidas na angiogénese. Descrita pela primeira vez em 1988,os princípios subjacentes a este ensaio são as células endoteliais que retêm a capacidade de dividir e migrar rapidamente, em resposta a sinais angiogénicos 11-13. Além disso, as células endoteliais são induzidas a diferenciar e formar estruturas semelhantes a tubos quando cultivadas numa matriz de extracto de membrana basal (BME). Estes tubos contêm um lúmen rodeado por células endoteliais ligadas entre si por meio de complexos juncionais. A formação do tubo ocorre rapidamente com a maioria dos tubos formando neste ensaio dentro de 2-6 horas, dependendo da quantidade e do tipo de estímulos angiogénicos. Vários tipos de células endoteliais pode ser utilizado para este ensaio, incluindo ambas as células primárias e as linhas de células imortalizadas 14,15. A linha celular utilizada para este artigo foi rato 3B-11, mas o mesmo método pode ser aplicado a outras linhas de células endoteliais, tais como SVEC4-10 (ratinho) ou células endoteliais primárias, tais como células humanas (HUVEC). Dependendo da linhagem de células é utilizado e se o ce endoteliallls são transformadas ou não transformadas, a optimização necessita de ser realizado para identificar o tempo ideal necessário para a formação do tubo adequado.

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			<td height="114" rowspan="2" style="height:114px;width:339px;">Costar 24-Well Tissue Culture-Treated Plate</td>
			<td rowspan="2" style="width:189px;">Corning</td>
			<td rowspan="2" style="width:124px;">3524</td>
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		<tr height="21">
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			<td height="190" rowspan="2" style="height:190px;width:339px;">BD Matrigel Basement membrane matrix, Growth factor reduced</td>
			<td rowspan="2" style="width:189px;">BD Biosciences</td>
			<td rowspan="2" style="width:124px;">354230</td>
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			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25300-054</td>
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			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">25030-081</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">15140-122</td>
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		<tr height="21">
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		<tr height="36">
			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DMEM (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">11965-092</td>
		</tr>
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		</tr>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco DPBS (1X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
			<td rowspan="2" style="width:124px;">14190-144</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Fetal Bovine Serum</td>
			<td rowspan="2" style="width:189px;">Atlas Biologicals</td>
			<td rowspan="2" style="width:124px;">F-0500-A</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Calcein AM</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:124px;">C3100MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMSO</td>
			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
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endothelial cell tube formation assay for the in vitro study of angiogenesis

A angiogénese é um processo fundamental para o desenvolvimento de tecido normal e cicatrização de feridas, mas também está associada a uma variedade de condições patológicas. Utilizando este protocolo, a angiogénese pode ser medida in vitro, de um modo rápido, quantificável. Células endoteliais primárias ou imortalizadas são misturados com meios condicionados e semeadas em matriz de membrana basal. As células endoteliais formam estruturas semelhantes a capilares em resposta a sinais angiogénicos encontrados em meios condicionados. A formação do tubo ocorre rapidamente com células endoteliais começam a alinhar-se dentro de 1 hora e túbulos contendo lúmen começando a aparecer dentro de 2 horas. Os tubos podem ser visualizados utilizando um microscópio invertido de contraste de fase, ou as células podem ser tratadas com calceína AM antes do ensaio e tubos visualizado através de fluorescência ou microscopia confocal. O número de locais de filiais / nós, laços / malhas, o número ou o comprimento dos tubos de formados podem ser facilmente quantificado como uma medida de em VITRo angiogénese. Em resumo, este ensaio pode ser usado para identificar genes e vias que estão envolvidos na promoção ou inibição de angiogénese de um modo rápido, reprodutível e quantitativa.

Rato 3B-11 as células endoteliais foram semeadas em factor de crescimento reduzida solidificada BME - neste ensaio, o produto foi utilizado Matrigel - e seguido ao longo do tempo. Como mostrado na Figura 1, os factores angiogénicos segregados por qualquer queratinócitos de ratinho ou de fibroblastos são capazes de induzir a formação do tubo ao longo do tempo. As células endoteliais migram e começam a se formar pequenos ramos dentro de 1-2 horas de galvanização. Formação máxima tubo foi alca...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

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

O ensaio de formao de tubo é um método rápido, quantificável para medir a angiogénese in vitro. As células endoteliais são combinados com meios condicionados e semeadas em extracto de membrana basal. A formação do tubo ocorre dentro de poucas horas e túbulos recém-formados facilmente quantificada.

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

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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

59.0K visualizações.  American University.  O ensaio de formao de tubo é um método rápido, quantificável para medir a angiogénese in vitro. As células endoteliais são combinados com meios condicionados e semeadas em extracto de membrana basal. A formação do tubo ocorre dentro de poucas horas e túbulos recém-formados facilmente quantificada. 

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

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

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: Um modelo in vitro para o Estudo da angiogênese (Entrevista)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

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 ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

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

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

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 ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Exteriorização Rat Mesentério: Um modelo para investigar a dinâmica celulares envolvidos na angiogênese

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

A quantificação da Adesividade celular endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

A Bioensaio simples para a avaliação dos fatores de crescimento endotelial vascular

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

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.

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

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.