Os autores gostariam de agradecer ao Dr. Peiman Hemmati (Departamento de Medicina da Universidade de Wisconsin-Madison) para oferecer generosamente as CTMs H1, e Dr. Hacker Tim, Dr. Gouqing Song e Ms. Jill Koch, da Universidade de Wisconsin Fisiologia Cardiovascular Facilidade núcleo para realização de cirurgias mouse. Este trabalho foi financiado pela National Science Foundation através de uma Bolsa de Investigação Pós-Graduação de Brian Freeman e R21 NIH HL089679.

1. Transfection célula doadora <ol><li> Células-tronco mesenquimais da colheita (MSCs derivadas de células-tronco embrionárias H1 gentilmente doados pelo Dr. Peiman Hematti, alternativamente, qualquer tipo de célula de qualquer espécie a hipótese de fundir in vivo pode ser empregado), quando 70-80% confluente com 1X tripsina (Mediatech, Manassas VA) por 5 min. Inativar tripsina com α-MEM médio completo (livre de antibióticos, Invitrogen, Carlsbad CA) 18. Centrifugar a 300 xg por 5 min. </li><li> Aspirar cuidadosamente o sobrenadante e re-suspensão pellet em 1 ml de 1X PBS e contagem de células usando um hemocitômetro. </li><li> Transferência de 1,5 x 10 6 células para um tubo de centrífuga de 1,5 mL. Centrifugar a 300 xg por 5 min. </li><li> Aspirar cuidadosamente o sobrenadante. Ressuspender pellet em 300 mL de R Buffer (Sistema de Transfection Neon, Invitrogen) e 6 mg (2 mg / 5,0 x 10 5 células) da P231 pCMVe-betaAc-STOP-luc (Addgene, Cambridge, MA). Colocar 3 mL de tampão de E (InvitrOgen) à porta docking eletroporação por protocolo do fabricante (Sistema de Transfection Neon, Invitrogen). </li><li> Transferência de células-plasmídeo solução para a ponta da pipeta de 100 L Neon e electroporate com uma duração de pulso de 20 ms e uma magnitude de 1,5 mil volts. Coloque electroporated células em um tubo cônico de 15 mL contendo 9,7 mL meio α-MEM completo. </li><li> Repita o passo 1,5 e duas vezes mais células transfectadas piscina para produzir um volume total de 10 mL. Adicione o 10 mL de suspensão celular (1,5 x 10 6 células) para um balão contendo 10 mL T175 α-MEM médio completo. Viabilidade celular após a eletroporação é de aproximadamente 30%, para produzir cerca de 4,5 x 10 5 células viáveis ​​por T175. </li><li> Alterar α-MEM completo médio de 24 horas após transfecção. </li><li> Colheita transfectadas células quando 70-80% confluentes (~ 2-3 dias após eletroporação). Realizar contagem de células usando um hemocitômetro e ressuspender as células em uma concentração de 1,0 x 10 6cells/50 mL de α-MEM médio completo. Células minimizar o tempo gasto em suspensão para reduzir a morte das células antes da injeção. </li></ol> 2. Injeção intramiocárdico <ol><li> Induzir a anestesia por isoflurano (Phoenix Pharmaceuticals, Inc., St. Joseph, MO) em camundongos manipulados geneticamente para expressar constitutivamente Cre recombinase em cada célula (B6.C-Tg (CMV-CRE) 1CGN / J, Jackson Laboratory, Bar Harbor, ME). </li><li> Remover os pêlos da região do tórax usando cortar cabelo ou removedor de pêlos químicos. </li><li> Entubar com um cateter de calibre 18 (Becton Dickinson &amp; Co, Franklin Lakes NJ) e colocar no ventilador do mouse no 120-130 respirações por minuto, com um volume de 150 mL acidente vascular cerebral. </li><li> Fazer incisão lateral em todo o quarto espaço intercostal produzindo assim uma toracotomia. </li><li> Visualizando o coração, fazer duas injeções 25 mL de suspensão de células transfectadas com uma seringa de 1 mL (Temuro Medical Corporation, Somerset, NJ) e agulha de calibre 28 (EBICTem Dickinson &amp; Co, Franklin Lakes NJ). Para facilitar a injeção intramiocárdica e evitar danos excessivos ao órgão, dobre a cabeça agulha ~ 90 graus. </li><li> Após a injeção, use suturas absorvíveis (por exemplo, vicryl) para fechar as costelas e as camadas musculares. Sutura de pele fechada com nylon ou seda 4-0. </li><li> Permitir mouse para recuperar da anestesia e extubação. </li><li> Grupos de controle devem incluir ratos Cre recebendo injeções único meio, ratos Cre receber a mesma concentração de células e camundongos untransfected tipo selvagem que receberam células transfectadas (alternativamente, os ratos que receberam células transfectadas Cre não propensas a fusível). </li></ol> 3. Imagem biofotônica <ol><li> De cinco a quinze minutos antes de imagem, por via intraperitoneal (IP) injetar 10 mL por grama de peso do mouse corpo de 15 mg / mL D-luciferina (Caliper Life Sciences, Hopkinton, MA). </li><li> Induzir a anestesia em camundongos através de isoflurano em 4% para a indução de umd 1 - 2% para a manutenção. </li><li> Camundongos lugar supina na caixa de imagem com máscara administração 1 - 2% de isoflurano para anestesia de manutenção (Imaging System Xenogen biofotônica, Hopkinton, MA). Vários ratos podem ser visualizados simultaneamente. Imagem do rato do grupo controle experimental com ratos para facilitar a comparação do sinal luminescente. </li><li> Usando software Vivendo Imagem (Xenogen), definir o tempo de exposição adequado (normalmente 60 segundos, ver Resultados). Definir área de imagem para caber ratos e manter consistente ao longo da área de imagem para evitar alterações na sensibilidade. Definir a altura sujeita a 4,5 cm. </li><li> Adquirir sinal de luminescência intensidade correspondente ao mouse ou ratos dentro do campo de visão e salvar arquivos de imagem não modificado. Imagens processo para remover sinal de fundo correspondente a um controle ou mouse não-manipulado. Valores de intensidade acima do fundo correspondem a células fundidas dentro do animal. Análise de intensidade pode ser realizado utilizando software Vivendo Imagem (Xenogen) ou open source software de análise de imagem. Typically, uma região de interesse (ROI) é selecionado para comparar os dados de intensidade entre os animais e experimentos. </li></ol> 4. Resultados representante Para determinar a sensibilidade do Sistema de Imagem Xenogen biofotônica, uma linha celular que expressa constitutivamente luciferase (231-LUC-D3H1, Xenogen) foi entregue para o miocárdio de ratos C57/Bl6 (Jackson Laboratory). Células foram injetadas em concentrações de 1 x 10 6, 1 x 10 3, ou 1 x 10 1 células. Seis horas após a entrega das células, os camundongos foram injetados intraperitonealmente com a luciferina e fotografada usando o sistema Xenogen. Um sinal específico pode ser detectado com 1.000 células (2 de 6 camundongos com imagens, Figura 2), mas a detecção é mais confiável, com 10.000 células (6 de 6 ratos com imagens). Importante, este estudo também serviu para estabelecer uma correlação aproximada entre o número de células que expressam luciferase e da intensidade do sinal. <p class="jove_content"&gt; Para demonstrar a utilidade do protocolo descrito na detecção e rastreamento de fusão celular, MSCs foram transfectadas com o plasmídeo LoxP-Stop-LoxP-luciferase (Addgene) e entregues para o miocárdio de Cre-expressando camundongos. Aproximadamente uma semana após o parto celular, as cobaias foram primeiro fotografada usando o sistema Xenogen sem D-luciferina injeção. Como esperado, sem o substrato enzimático, nenhum sinal foi detectado intensidade (Figura 3). Em seguida, D-luciferina foi injetada por via intraperitoneal e um sinal correspondente a intensidade luciferase e, portanto, fusão celular foi detectado em duas das quatro camundongos testados. Um sinal semelhante foi detectado uma semana depois (Figura 3), sugerindo produtos MSC-coupled de fusão pode ser mantida in vivo. Neste caso, o estudo foi interrompido para permitir a avaliação de coração e de tecido circundante, mas pode-se imaginar a longo prazo análises e imagens mais freqüentes para acompanhar a manutenção proliferação, umad migração, talvez, de produtos de fusão em camundongos. <img alt="Figura 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Figura 1. Esquemático da técnica para detectar a fusão celular in vivo. Se a fusão entre Cre-expressando células de camundongo e células transplantadas expressando um plasmídeo floxed luciferase ocorre, luciferase será expressa. Luciferase podem ser detectados através da injeção do substrato enzimático, D-luciferina, para o rato e, em seguida, imagens do mouse através de um Sistema de Imagem Xenogen biofotônica (Adaptado de 19) <img alt="Figura 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Figura 2. Sensibilidade de detecção de luciferase células que expressam no tecido cardíaco com imagens biofotônica. A linha de células que constitutivamente expressa luciferase (231-LUC-D3H1, Xenogen) foi entregue ao espaço intramiocárdico de C57/Bl6 ratos em vários números de células total. Imagens representativas dos camundongos retentes para perceber 1 x 10 6, 1 x 10 3 e 1 x 10 1 células (da esquerda para a direita) são mostrados, a imagem foi conduzida aproximadamente 6 horas após a injeção. <img alt="Figura 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Figura 3. Na quantificação da luminescência Vivo indicativa de fusão celular. MSCs foram transfectadas com o LoxP-Stop-LoxP-luciferase plasmídeo e entregue para o miocárdio de Cre-expressando camundongos. Aproximadamente uma semana e duas semanas após o parto celular, ratos Cre foram fotografadas usando o Sistema de Imagem Xenogen biofotônica para medir a intensidade da luminescência indicativo de fusão celular. (A) Sobreposição de fotografia e intensidade da luminescência de farsa e camundongos 1-4 (esquerda para a direita) dias após o parto 17 células. (B) A intensidade da luminescência de farsa e camundongos 1-4 (esquerda para a direita) dias após o parto 17 células. A região de interesse foi selecionada (amarelo) correspondente ao local da injeção e intensidade levels foram determinados utilizando software ImageJ (fonte livre) 20. (C) A intensidade da luminescência foi normalizado para a mesma região de interesse na farsa do mouse para todas as condições experimentais. Menos uma semana, 3 e 4 ratos mostrou sinal de luminescência positivos sugerindo a fusão espontânea de uma célula do rato e MSC transplantado. O sinal persistiu em mouse de 3 em duas semanas. Para determinar a localização de órgãos específicos do sinal correspondente ao mouse de 3, a cavidade torácica foi exposto e principais órgãos extirpados e imagens. (D) Sobreposição de fotografia e intensidade da luminescência do mouse 3. Localização nota de intensidade de sinal no intestino delgado. (E) A intensidade da luminescência do mouse 3. (F) sobreposição de fotografia e intensidade da luminescência da farsa mouse. (G) A intensidade da luminescência de sham mouse.

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O método descrito aqui permite, pela primeira vez, a identificação discreta e análise temporal da fusão celular em organismos, incluindo animais de pequeno porte. A abordagem combina Cre-LoxP recombinação com análise de imagem subseqüentes biofotônica. A abordagem é passível de rastreamento não só a fusão célula-célula, mas também do vírus na célula-fusion e assim pode ser útil para rastrear infecções virais. Análise de imagem é rápida e é possível de várias imagens de pequenos animais simul...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Neon Sistema Transfection </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Kit Neon 100 L </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Contém R e E do buffer </td></tr><tr><td> a-MEM em pó </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> De soro fetal bovino (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-CRE) 1CGN / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> Tripsina 10X </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25-054-Cl </td><td></td></tr><tr><td> L-Glutamina </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-Luciferina </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Imaging System biofotônica </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> Spectrum IVIS </td><td></td></tr><tr><td> Biocarbonate de sódio </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Não-essenciais Aminoácidos </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

A capacidade de duas ou mais células do mesmo tipo para fundir tem sido utilizado em metazoários ao longo da evolução para formar muitos órgãos complexos, incluindo o músculo esquelético, ossos e placenta. Estudos contemporâneos demonstram fusão de células do mesmo tipo confere função melhorada. Por exemplo, quando as células do trofoblasto do fusível placenta para formar o sinciciotrofoblasto, o sinciciotrofoblasto é mais capaz de transportar nutrientes e hormônios através da barreira materno-fetal do trofoblasto não fundido 1-4. Estudos mais recentes demonstram a fusão de células de diferentes tipos pode direcionar o destino da célula. A &quot;reversão&quot; ou modificação do destino da célula por fusão já foi pensado para ser limitada a sistemas de cultura de células. Mas o advento do transplante de células-tronco levou à descoberta por nós e outras que as células-tronco podem se fundir com as células somáticas in vivo e que a fusão facilita a diferenciação de células-tronco 5-7. Assim, fusão celular é um processo regulado capable de promover a sobrevivência e diferenciação celular e, portanto, poderia ser de importância central para o reparo, o desenvolvimento de tecidos e até mesmo a patogênese da doença. Limitar o estudo da fusão celular, é a falta de tecnologia apropriada a 1) identificar com precisão produtos de fusão e 2) produtos de fusão faixa ao longo do tempo. Aqui apresentamos uma nova abordagem para abordar tanto limitações através da indução de bioluminescência em fusão (Figura 1);. Bioluminescência pode ser detectado com alta sensibilidade in vivo 15/08 Nós utilizamos uma construção que codifica a luciferase do firefly (Photinus pyralis) gene colocado adjacente ao um códon de parada flanqueado por seqüências LoxP. Quando as células expressando este gene fusível com células expressando a proteína Cre recombinase, os sites são clivados e LoxP o sinal de parada é retirado permitindo a transcrição de luciferase. Como o sinal é inducible, a incidência de falsos-positivos sinais é muito baixa. Ao contrário dos métodos já existentes, que utilizam o sistema Cre / LoxP 16, 17, foi incorporado um &quot;viva&quot; sinal de detecção e, assim, pagar pela primeira vez a oportunidade de acompanhar a cinética de fusão celular in vivo. Para demonstrar a abordagem, os camundongos ubiquitously expressar Cre recombinase serviu como receptores de células-tronco transfectadas com uma construção para expressar a jusante luciferase de um códon de parada floxed. Células-tronco foram transplantadas via injeção intramiocárdica e depois do transplante de análise de imagem intravital foi conduzido para rastrear a presença de produtos de fusão no coração e tecidos circundantes ao longo do tempo. Esta abordagem pode ser adaptada para analisar fusão celular em qualquer tipo de tecido, em qualquer estágio da doença, desenvolvimento ou reparação de tecidos adultos.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Um método para controlar a fusão de células de organismos vivos ao longo do tempo é descrito. A abordagem utiliza Cre-<em&gt; LoxP</emRecombinação&gt; para induzir a expressão luciferase sobre fusão celular. O sinal luminoso gerado pode ser detectado nos organismos vivos utilizando sistemas de imagens biofotônica com uma sensibilidade de detecção de ~ 1.000 células nos tecidos periféricos.

A Cre-Lox Abordagem Recombinação P para Detecção de fusão celular In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

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JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K visualizações.  University of Wisconsin-Madison. Um método para controlar a fusão de células de organismos vivos ao longo do tempo é descrito. A abordagem utiliza Cre- LoxP para induzir a expressão luciferase sobre fusão celular. O sinal luminoso gerado pode ser detectado nos organismos vivos utilizando sistemas de imagens biofotônica com uma sensibilidade de detecção de ~ 1.000 células nos tecidos periféricos.

Vídeo: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Detecção de Biomolecular empregando o sensor de imagem Interferometric Reflectância (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Bioengenharia

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricação de Eletroquímica-DNA Biossensores para Detecção Reagentless de ácidos nucléicos, proteínas e pequenas moléculas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Criando Two-Dimensional Substratos modelado para Confinamento de proteína e celular

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simples dispositivos microfluídicos para<em&gt; In vivo</em&gt; Imaging de<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; E Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinjeção Ferida Ensaio e<em&gt; In vivo</em&gt; Localização de Epidermal Wound resposta Jornalistas em<em&gt; Drosophila</em&gt; Os embriões.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

A pele completa Defeito Modelo Estudar a vascularização de Biomateriais<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

A técnica de livre-Label para a imagem espaço-temporal do Secreções única célula

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Biorreactores de fibras ocas para<em&gt; In Vivo</em&gt; -como Cultura tecidos de mamíferos

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparação de 3D colágeno Géis e microcanais para o estudo das interações 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Detecção visual de vários ácidos nucleicos em uma matriz capilar

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...