DIY criogênico dispositivo de recuperação tinha sido testado no decorrer dos projetos &quot;Modelagem prenilação proteína bacteriana em células eucarióticas utilizando abordagens moleculares e de bioinformática&quot;, patrocinado pelo Prêmio Mary Louise Andrews para o programa de Pesquisa do Câncer, Virginia Academy of Science &quot;, significado fisiológico da edição de RNA no Plasma Células dendríticas &quot;, patrocinado pela F. Thomas Jeffress e Kate Miller Jeffress Memorial Trust e Contrato 16.740.11.0364 Estado (Ministério da Ciência e Educação, Rússia). Gostaríamos de agradecer a Beth Eom para obter ajuda com o experimento integridade do RNA.

1. Montagem do dispositivo Cryotolerant para Recuperação de Cryoboxes <ol><li> Usando um alicate, endireitar um lado da face 3 Nota uma forte L-tie (Figura 1A) para fazer forte-tie em forma mostrada na Figura 1B:. As dimensões do tie-forte pode ser seleccionada dependendo do diâmetro do gargalo do Dewar . </li><li> Garantir dois tão fortes de laços com uma cinta de T-tie forte (Figura 2) usando Crown porca e parafuso, anilha e parafuso para formar a base Cryoscoop como mostrado na Figura 3 (AB). Nota: Dois fortes são usadas para permitir a recuperação de 5 ½ x 5 ½ pol cryoboxes. </li><li> Utilizando uma placa ranhurada (Figura 4), ​​alinhar as ranhuras na cinta forte-tie T com as ranhuras em uma extremidade da placa ranhurada e fixar os dois em conjunto com a ajuda de Hillman porca e parafusos (Figura 5). Nota: Se o placa entalhada e forte laço de não come com furos pré-perfurados, você terá que ter furos perfurados utilizando carboneto de broca. </li><li> Dobrar a outra extremidade da placa ranhurada com a ajuda de um alicate, como mostrado na Figura 6. (Este passo é opcional). </li><li> Enrole a alça com espuma para compreender melhor o dispositivo quando em azoto líquido, como mostrado na Figura 6. (Opcional) Nota: Remova a espuma antes da autoclavagem. </li></ol> 2. Montagem do dispositivo Cryotolerant para Recuperação de criotubos O dispositivo de recuperação Cryotolerant DIY pode também ser adaptado para a recuperação de criotubos. Isto envolve a substituição da base plana, com um filtro de aço inoxidável como mostrado alterados para escavar para fora a partir de frascos de azoto líquido (Figura 7). <ol><li> Soltar a porca da Coroa e os parafusos que prendem a forte-tie alça T para a placa entalhada. </li><li> Antes de fixar o filtro de aço inoxidável à placa ranhurada, dobre o pescoço doo filtro com a ajuda de um alicate para orientar a base com um ângulo para se assemelhar a uma concha (Figura 8) Nota: Se o filtro é fornecido com um lábio, usar um alicate para dobrar o lábio para baixo, o que irá fazer com a manobra mais fácil do dispositivo.. </li><li> Alinhe o orifício na alça do filtro junto com os furos na placa entalhada e proteger o filtro com a ajuda de porcas e parafusos Hillman como mostrado na Figura 8. Nota: se o filtro não vem com furos pré-perfurados, você vai precisar de arranjar para ter furos perfurados com broca de carboneto. </li></ol> 3. Recuperando Cryoboxes e criotubos De Dewar <ol><li> Para começar, coloque luvas criogênicas e remover 1 ou 2 prateleiras de resfriamento do Dewar para permitir espaço para manobras. </li><li> Raspar suavemente ao longo da parte inferior do Dewar com o dispositivo de recuperação Cryotolerant DIY vertical equipado com forte-tie base de cinta T (Figura 5), e recolher o falle caixan para o fundo do tanque Nota Dewar:. ferve de azoto líquido imediatamente em contacto com um objeto mais quente, que envolve o objecto no gás isolante azoto (efeito Leidenfrost). Para arrefecer o dispositivo antes da recuperação de amostras, lentamente imergir o dispositivo no Dewar e permitir azoto líquido para arrefecer o dispositivo suficientemente antes de prosseguir com o passo acima. </li><li> Puxar o dispositivo na posição vertical ao longo das paredes do Dewar para colher a caixa congelador para fora do tanque. </li><li> Agarrar a caixa recuperados a partir do fundo do Dewar. </li><li> Para recuperar criotubos, raspar suavemente a parte inferior da Dewar com o cryoscoop equipado com um filtro (Figura 9). </li><li> Semelhante ao cryobox recuperação, puxe o dispositivo na posição vertical e pegue os frascos coletados no coador. </li></ol> 4. Os resultados representativos Um grande desafio em estudos de expressão de genes é que a qualidade do RNA depende das condições de armazenamentopara ambas as amostras de tecido congelado e Figura extraída RNA 4. 10 mostra o efeito da manipulação da amostra sobre a integridade do RNA total. O RNA total a partir da fonte de tecido mesmo (adiposo humano) foi submetido a manipulação da amostra diferente. Antes da experiência, intacta RNA total preparate mostra claras bandas 28S e 18S rRNA com a banda rRNA 28S aproximadamente duas vezes tão intensa como a banda de rRNA 18S (Figura 10, pista 1). Esta proporção de 2:1 (28S: 18S) é uma indicação de que o RNA está intacta, enquanto que o RNA parcialmente degradadas aparece como uma mancha, ou a sua 28S: 18S relação da intensidade de partida de 2:01 5. As pistas 3-4 descreve amostras de RNA com uma degradação significativa. Estas amostras de RNA foram sujeitas a repetidos descongelamento parcial, por curto prazo consultas das Dewar (Método A), assim modelando o armazenamento a longo prazo com periódica decantação, devido à necessidade de a recuperação do frascos caído e caixas (Figura 10, Lane 3-4 ). No entanto, as amostras armazenadas em Dewar atendidousando DIY dispositivo (Método B) e, portanto, não descongelamento parcial, contêm RNA intactas (Figura 10, pista 2). <img alt="A Figura 1" src="/files/ftp_upload/3903/3903fig1.jpg" /> Figura 1. A) A base é constituído por duas faces 3 forte-tie de dimensões indicadas. Cada ligação forte contém um furo pré-perfurado grande (seta sólida) e múltiplos orifícios mais pequenos (seta pequena) em cada face. Isso fornece uma opção para permitir a fixação com porca e parafusos. Além disso, os orifícios mais pequenos permitir a drenagem rápida de azoto líquido durante a recuperação de amostra. B) Um lado de cada um dos laço forte-é endireitado com a ajuda de um alicate para fazer em forma de L-tie forte. <img alt="A Figura 2" src="/files/ftp_upload/3903/3903fig2.jpg" /> Figura 2. Forte-tie alça T é usado para proteger os fortes laços de conjunto. Como forte-tie, T-cinta tem um furo pré-grandes (seta contínua) e dois furos menores (small seta) em cada canto. Isto permite garantir forte-dades ao longo do comprimento do T - Strap usando porcas e parafusos. Também permite a fixação T-cinta para a placa ranhurada. <img alt="A Figura 3" src="/files/ftp_upload/3903/3903fig3.jpg" /> Figura 3. A) Cada laço forte-é presa a um canto de T-cinta, ao longo do seu comprimento com porcas de parafuso de coroa, parafusos e anilhas. B) Dois forte-dades são protegidos adjacentes uns aos outros para ajudar a recuperar 5 ½ polegadas x 5 ½ dentro cryoboxes. Para cryoboxes menores, como 2 polegadas x 2 polegadas, um único forte-tie pode ser fixado diretamente à placa entalhada sem cinta-T. <img alt="A Figura 4" src="/files/ftp_upload/3903/3903fig4.jpg" /> Figura 4. Ranhurada placa plana é utilizada como a alça. Comprimento da placa ranhurada depende da profundidade do Dewar. Neste caso, 4 pés de placa ranhurada foi usado. A espessura da placa ranhurada é s críticoesde finas placas de fenda (&lt;14 medidores) fazer o dispositivo frágil ao recuperar amostras de nitrogênio líquido cheio Dewar. <img alt="A Figura 5" src="/files/ftp_upload/3903/3903fig5.jpg" /> Figura 5. A base plana constituída por fortes-dades e cinta T é então fixada a uma extremidade da placa ranhurada. O orifício maior no canto restante da correia T está alinhada com a ranhura na placa plana e fixado utilizando o parafuso e porca sextavada. Este dispositivo pode ser agora usado para recuperar cryoboxes de Dewar. <img alt="A Figura 6" src="/files/ftp_upload/3903/3903fig6.jpg" /> Figura 6. Uma extremidade da placa ranhurada plana é dobrada com um alicate e coberto com espuma para proporcionar uma melhor aderência especialmente para Dewars profundas, como mostrado no presente caso. <img alt="A Figura 7" src="/files/ftp_upload/3903/3903fig7.jpg" /> Figura 7. Filtro com malha de arame e cabo de aço inoxidávelé usado para recuperar criotubos de Dewar. Se o identificador não está provida de uma ranhura, perfurar com bit carboneto será necessário para a perfuração de um orifício. <img alt="A Figura 8" src="/files/ftp_upload/3903/3903fig8.jpg" /> Figura 8. Para facilitar a recuperação de criotubos, filtro é dobrado ao longo do pescoço usando alicates para obter filtro de concha em forma. Se o filtro está provido de lábio como no presente caso, alicates pode ser usado para dobrar. Isso garante o filtro não fica presa dentro do Dewar. <img alt="A Figura 9" src="/files/ftp_upload/3903/3903fig9.jpg" /> A Figura 9. Filtro é fixada à placa ranhurada plana numa extremidade usando porca sextavada e parafuso. <img alt="A Figura 10" src="/files/ftp_upload/3903/3903fig10.jpg" /> Figura 10. Integridade da amostra de RNA total submetido a diferentes condições de manejo da amostra. Para explorar como a qualidade do RNA é afetado pela manipulação da amostra cONDIÇÕES, um conjunto de amostras de ARN a partir da mesma fonte foi submetido a dois métodos de tratamentos. Método A consistiu de armazenamento de RNA em Dewar submetido a recuperações de exemplo usando dispositivo DIY, portanto, não descongelamento parcial. Método B consistiu de armazenamento de RNA em Dewar submetido a repetidas descongelamento parcial, por curto prazo consultas das Dewar, assim modelando o armazenamento a longo prazo com periódica decantação para recuperar amostras caídas. Integridade de RNA total foi então analisada por electroforese em gel. Lane1: Amostra antes do armazenamento em Dewar; Pista 2: Amostra de Dewar submetido a recuperação pelo método A; Pista 3, 4: Exemplo de Dewar submetido a recuperação pelo método B, M: 100 pb ladder.

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	<li>Kittel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cryogenic Engineering. , 41 (1996).</li><li>Edeskuty, F. J., Walter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stewart Safety in the handling of cryogenic fluids. , (1996).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. , (2011).</li><li>Muyal, J., Muyal, V., Kaistha, B., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutation Research/Reviews in Mutation Research. 543, 217-234 (2003).</li><li>Lynch, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CELL,+TISSUE+AND+ORGAN+CULTURE+|+Storage+and+Cryopreservation.">CELL, TISSUE AND ORGAN CULTURE | Storage and Cryopreservation.</a> Encyclopedia of Rose Science. , 106-111 (2003).</li><li>Crawford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics Engineering. , (1998).</li><li>Gorenc, B., Tinyou, R., Syam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Steel designers' handbook. , (1996).</li><li>Harper, C. A., Petrie, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+materials+and+processes:+a+concise+encyclopedia.">Plastics materials and processes: a concise encyclopedia.</a> Wiley-Interscience. , (2003).</li></ol>

Não há conflitos de interesse declarados.

Um dos métodos mais comuns de recuperar cryoboxes e criotubos a partir do fundo do tanque Dewar é decantar azoto líquido para um recipiente de reserva e puxar para fora as amostras restantes no balão ou flutuando na azoto líquido decantado. Isto é, no entanto, uma prática insegura que pode provocar queimaduras criogénicos ou asfixia devido a exposição prolongada a vapores de azoto líquido 2. Outra forma comum de recuperação de criotubos caídos envolve o uso de pinças criotubo 3. No e...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Hillman Grupo Plated Slotted planas 1-3/8 x 4 pés; calibre 14 </td><td> Loja de ferragem local </td><td> N / A </td><td> 1 unidade necessária </td></tr><tr><td> Coroa Parafuso de aço inoxidável # 6 - 32 x 3/8 pol Oval Cabeça Parafuso Unidade Phillips </td><td> Loja de ferragem local </td><td> Modelo # 42281 </td><td> 4 unidades necessárias </td></tr><tr><td> Coroa Parafuso 3/8 pol x 1-1/2 pol zincado Arruelas de Aço Fender </td><td> Loja de ferragem local </td><td> Modelo # 20122 </td><td> 4 unidades necessárias </td></tr><tr><td> Coroa Parafuso galvanizado # 8-32 Nut Parafuso </td><td> Loja de ferragem local </td><td> Modelo # 18512 </td><td> 4 unidades necessárias </td></tr><tr><td> Simpson Strong-Tie BC40Z 4 In.x4 dentro Metade da Base </td><td> Hardware localfazer compras </td><td> Modelo # BC40Z </td><td> 2 unidades necessárias </td></tr><tr><td> 6 Simpson Strong-Tie pol x 6 pol Correia T </td><td> Loja de ferragem local </td><td> Modelo # 66T </td><td> 1 unidade necessária </td></tr><tr><td> Oneida 6 &quot;peneira </td><td> Supermercados local </td><td> Modelo # 7859W </td><td> 1 unidade necessária </td></tr><tr><td> O Grupo Hillman Galvanizado Nuts Hex 3/8-16 </td><td> Loja de ferragem local </td><td> Modelo #: 810,509 </td><td> 6 unidades necessárias </td></tr><tr><td> O Grupo Hillman Galvanizado parafusos sextavados 3/8- 16 </td><td> Loja de ferragem local </td><td></td><td> 4 unidades necessárias </td></tr><tr><td> Espuma para isolamento </td><td> Loja de ferragem local </td><td></td><td> 1 unidade necessária </td></tr><tr><td> Chave de fenda </td><td> Loja de ferragem local </td><td></td><td> 1 unidade necessária </td></tr><tr><td> Alicate </td><td> Loja de ferragem local </td><td></td><td> 1 unidade necessária </td></tr></tbody></table>

do-it-yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

O nitrogênio líquido é incolor, inodoro, extremamente frio (-196 ° C) líquido mantido sob pressão. É comummente usado como um fluido criogénico para armazenamento a longo prazo de materiais biológicos tais como sangue, células e tecidos 1,2. A natureza criogénico de azoto líquido, enquanto ideal para a preservação da amostra, pode causar congelação rápida dos tecidos vivos em contacto - conhecido como &#39;criogénico queimadura&#39; 2, o que pode levar a queimaduras graves em pessoas intimamente envolvidas no armazenamento e recuperação de amostras de Dewars. Além disso, como o azoto líquido evapora que reduz a concentração de oxigénio no ar e pode causar asfixia, especialmente em espaços confinados 2. Nos laboratórios, amostras biológicas são frequentemente armazenados em criotubos ou cryoboxes empilhados em prateleiras de aço inoxidável dentro dos tanques de Dewar 1. Estes suportes de armazenamento são fornecidos com um eixo de tempo para impedir caixas de deslizar para fora dos suportes e na parte inferior doDewars durante o manuseio de rotina. Com demasiada frequência, no entanto, caixas ou frascos com amostras preciosas escorregar e afundar até o fundo do tanque de nitrogênio líquido preenchido. Em tais casos, as amostras podem ser tediosamente recuperada após a transferência do azoto líquido para um recipiente de reserva ou descartá-lo. As caixas e frascos pode então ser recuperado a partir com relativa segurança Dewar esvaziado. No entanto, a natureza criogénico de azoto líquido e da sua taxa de expansão torna perigoso recuperação afundado amostra. É geralmente recomendado pelo Escritórios de segurança que a recuperação da amostra nunca ser realizada por uma única pessoa. Outra alternativa é usar comercialmente disponíveis grabbers frescos ou pinças para retirar os 3 frascos. No entanto, a visibilidade limitada dentro dos escuros cheios de líquidos Dewars representa uma grande limitação na sua utilização. Neste artigo, descrevemos a construção de um dispositivo Cryotolerant recuperação DIY, que faz recuperação de amostra de Dewar contendo fluidos criogênicos tanto a segurança de umnd fáceis.

Watch this Scientific Journal Video about Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks at JoVE.com

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

Aqui nós apresentamos um baixo custo, durável cryotolerant dispositivo para a recuperação de amostra de Dewar tanques cheios de nitrogênio líquido. A facilidade de construção e design modular do dispositivo faz com que o processo de recuperação da amostra de tanques criogênicos seguro e fácil.

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 ...

do-it-yourself-device-for-recovery-cryopreserved-samples-accidentally

Research

JoVE Journal

Biology

25.2K visualizações.  George Mason University. Aqui nós apresentamos um baixo custo, durável cryotolerant dispositivo para a recuperação de amostra de Dewar tanques cheios de nitrogênio líquido. A facilidade de construção e design modular do dispositivo faz com que o processo de recuperação da amostra de tanques criogênicos seguro e fácil.

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

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabricação de um dispositivo micro para a compartimentação de Neuron Soma e axônios

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biologia

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

Um dispositivo micro com padrões Groove para estudar o comportamento celular

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Um dispositivo de geração de Gradient microfluídicos de Biologia Celular

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Preparando E18 neurônios de rato para Cortical Compartimentação em um dispositivo micro

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

As amostras de teste para otimizar STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomic análise de Salmonella de alimentos e amostras ambientais

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

Técnicas reprodutivas para monitoramento e controle ovariano em anfíbios

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Amostra criogênica carregando em um espectrômetro de ressonância magnética nuclear de ângulo mágico que preserva a viabilidade celular

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.