Este estudo foi financiado pelo National Health and Medical Research Council of Australia (projeto de subsídio não. 6.288.480) e os Scobie e Clare MacKinnon Trust.

1. Micromixing uma reação RT <ol><li> Antes de executar uma reação de RT com micromixing, equilibrar a micromisturador para a temperatura desejada da reação de RT. <ol><li> Coloque o micromisturador dentro de um 37 ° C (ou a temperatura recomendada pelo fornecedor RT) incubadora de pelo menos 1 hora antes do início da reação de RT. </li></ol></li><li> Configure o mix RT de acordo com a transcriptase reversa do fornecedor (por exemplo, MMTV-RT da Promega, Omniscript da Qiagen) instruções, excepto o uso de uma única célula quantidades de RNA total de entrada (por exemplo, 0.1-1pg/μl), ao invés do g-ng quantidades recomendadas pelos fornecedores. <ol><li> Usamos padrão estéril, livre de nuclease 200μL tubos de paredes finas PCR. </li></ol></li><li> Posição tubos RT firmemente na micromisturador assim que estão prontos para a mistura. O micromisturador deve permanecer dentro da incubadora de 37 ° C para a duração da reação de RT. </li><li> Selecione a freqüência adequada e amplitude de micromixing. Neste exemplo, vamos usar 150Hz. </li><li> Mudar o micromisturador sobre e deixe-a por toda a duração da reação de RT (60 minutos). </li><li> Mudar o micromisturador e levar os tubos RT uma vez a reação RT está completa. </li><li> Coloque os tubos no gelo RT e prossiga normalmente com outras aplicações, tais como Reação em Cadeia da Polimerase quantitativa (qPCR ou PCR em tempo real). </li></ol> 2. Resultados representativos: O benefício de micromixing no contexto de reações RT é um aumento no rendimento em relação ao cDNA quando outros métodos de mistura (por exemplo, agitação, vórtex ou trituração) são utilizados. Isto pode ser visto, por exemplo, na análise subseqüente do cDNA Reação em Cadeia da Polimerase utilizando quantitativa (qPCR ou PCR em tempo real). Figura 2A ilustra os efeitos da micromixing para os primeiros cinco minutos (azul, &quot;micromixed5&quot;) ou a totalidade de 60 minutos (vermelho &quot;, micromixed60&quot;) em comparação com a trituração da reação RT imediatamente anterior à incubação RT (preto, &quot;trituração&quot; ). Neste caso o volume da reação de RT anterior foi 25μL e continha 2.5pg de RNA total. traços qPCR usando oligonucleotídeos desenhados para detectar cDNA representando Nurr-1 mRNA são mostrados acima, e os meios ± SEMs do número de ciclos para chegar a fluorescência máxima de 50% em três repetições deste mesmo experimento são plotados abaixo. O asterisco representa uma diferença estatisticamente significativa (one-way ANOVA com comparações múltiplas de Tukey). Note-se que micromixing para os primeiros cinco minutos da reação RT anterior produziu uma melhoria não significativa mais de trituração enquanto micromixing para toda a 60 minutos da reação RT anterior produziu uma melhoria significativa sobre a trituração, a magnitude do que foi equivalente a cerca de 15 qPCR ciclos, em média. O benefício de micromixing também pode ser visto na análise da curva de fusão dos produtos qPCR subseqüentes obtidos. No experimento ilustrado na Figura 2B, micromixing para toda a 60 minutos da reação RT anteriores resultaram em um sinal consistente qPCR em amostras duplicadas (Figura 2BA, 2 traços vermelhos), enquanto micromixing para os primeiros 5 minutos ou trituração só produziu qPCR inconsistentes traços (Figura 2BA, 2 traços azuis e dois traços negros, respectivamente). Os traços cinza são controles negativos (&quot;neg.&quot;) Na qual cDNA foi deixado de fora da reação qPCR. Quando os produtos qPCR deste experimento foram desnaturados por ramping sua temperatura (isto é, análise da curva de fusão) era evidente que certos produtos PCR (ou seja, um amplicon que era idêntico ao obtido quando as concentrações muito maiores de mRNA foram transcritas e reverter amplificado pela qPCR, dados não mostrados) estavam presentes apenas reações seguintes RT que tinha sido micromixed por 60 minutos (Figura 2BB, 2 traços vermelhos). Todos os outros (isto é, micromixed5, trituração e controles negativos) gerados produtos amplicon alternativo (por exemplo, dímeros primer). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de fluxo do protocolo para aplicação micromixing a reações padrão de laboratório RT. Descrições mais detalhadas sobre o princípio físico envolvido, a micromisturador e como construir um micromisturador in-house são fornecidos nas referências 13,14 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Figura 2"/> Figura 2. Dados Representante qPCR ilustrando os benefícios da micromixing aplicado a reações anteriores RT composto de uma única célula quantidades de mRNA em outra forma padrão de reações de laboratório RT. Vestígios Um Exemplo qPCR detectar mRNA representando cDNA que codifica a Nurr-1 gene. As reações anteriores RT contidas 2.5pg de RNA total em um volume de 25μL e foram realizadas em uma incubadora a 37 ° C por 60 minutos. RT reações foram misturados por trituração do RT misturar antes de incubação (traços pretos &quot;, trituração&quot;), micromixed durante os primeiros cinco minutos da reação de RT (traços azuis &quot;, micromixed5&quot;) ou micromixed para toda a 60 minutos da reação de RT (traços vermelhos &quot;, micromixed60&quot; ). Médias ± SEMs do número de ciclos de qPCR necessária para alcançar a fluorescência máxima de 50% são plotados abaixo para n = 3 repetições deste experimento. Asterisco indica uma diferença significativa (one-way ANOVA com comparações múltiplas de Tukey). Experimento B Outra qPCR examinar Nurr-1 produto cDNA, desta vez seguintes reações RT contendo 25pg de RNA total em um volume de 25μL. traços qPCR (a), bem como análise da curva de fusão dos produtos obtidos qPCR (b) são mostradas para as diferentes condições de mistura. Controles negativos em que nenhum cDNA foi incluído na reação de RT anteriores também estão incluídos (traços cinza &quot;, neg.&quot;). RT reações com concentrações muito maiores de mRNA também foram realizadas e produziram picos de fusão curva (dados não mostrados) idênticos aos (b) para micromixed60 (traços vermelhos) amostras.

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	<li>Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+Funct+Genomic+Proteomic.">Brief Funct Genomic Proteomic.</a> 2 (1), 31-31 (2003).</li><li>Aumann, T. D., Gantois, I., Egan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Exp Neurol. 213 (2), 419-419 (2008).</li><li>Ottino, J. M., Wiggins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos Transact A Math Phys Eng Sci. 362 (1818), 923-923 (2004).</li><li>Losey, M. W., Jackman, R. J., Firebaugh, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Microelectromech. Syst. 11, 709-709 (2002).</li><li>Bontoux, N., Dauphinot, L., Vitalis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab Chip. 8 (3), 443-443 (2008).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. 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> Anal Chem. 78 (9), 3084-3084 (2006).</li><li>Marcus, J. S., Anderson, W. F., Quake, S. 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> Anal Chem. 78 (3), 956-956 (2006).</li><li>Warren, L., Bryder, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc Natl Acad Sci U S A. 103 (47), 17807-17807 (2006).</li><li>Jiao, Z. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 6, 847-847 (2009).</li><li>Ahmed, D., Xiaole, M., Juluri, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microfluidics Nanofluidics. 7, 727-727 (2009).</li><li>Paxton, W. F., O'Hara, M. J., Peper, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 80, 4070-4070 (2008).</li><li>Autom, L. a. b. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 11, 399-399 (2006).</li><li>Petkovic-Duran, K., Manasseh, R., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 47 (4), 827-827 (2009).</li><li>Boon, W. C., Petkovic-Duran, K., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 50 (2), 116-116 (2011).</li><li>Liu, R. H., Lenigk, R., Druyor-Sanchez, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anal Chem. 75 (8), 1911-1911 (1911).</li><li>Riley, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ann+Rev+Fluid+Mech.">Ann Rev Fluid Mech.</a> 33, 43-43 (2001).</li><li>Whitehill, J., Neild, A., Ng, T. 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> Applied Physics Letters. 96 (5), 053501-053501 (2010).</li></ol>

Os autores não têm nada a revelar.

O método de aplicação do micromixing a reações padrão de laboratório RT descrito aqui pode, evidentemente, envolvem mRNA colhidas através de qualquer método (por exemplo, células de lise, microdissecção de captura de laser). Pode também incluir quaisquer marcas ou tipos de reagentes RT, qualquer temperatura (dentro da tolerância dos materiais do micromisturador), e qualquer período de tempo. Por exemplo, temos observado melhoria rendimentos cDNA a partir de reações RT compreendendo hexâmero aleatória ...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> RNA total foi isolado de pressão agudamente congelados preparados fatias mesencéfalo adulto do rato </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA Isolation Kit </td><td> Arcturus, CA, EUA </td><td> KIT0204 </td><td> O kit já está disponível na Applied Biosystems </td></tr><tr><td> DNA livre de DNase Tratamento e Reagentes Remoção </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Aleatória hexâmero primers </td><td> Promega </td><td> C1181 </td><td></td></tr><tr><td> AMV-RT </td><td> Promega </td><td> M5101 </td><td></td></tr><tr><td> dNTP set </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasin Inibidor ribonuclease </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Nuclease água livre de </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Green PCR Master Mix </td><td> Applied Biosystem </td><td> 4309155 </td><td></td></tr><tr><td> HPRT frente (20mer): CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> HPRT reversa (20mer): TAT CCC GTC TGA CGT CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 frente (23mer): CAG CTC TTT CGA CTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 reversa (23mer): GAG GGT CAT GTC GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 Espectrofotômetro. </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Corbett Rotor Gene RG-6000 </td><td> Corbett Life Science </td><td></td><td> Corbett Life Science foi adquirida pela QIAGEN em Julho de 2008 </td></tr></tbody></table>

increasing cdna yields from single-cell quantities of mrna in standard laboratory reverse transcriptase reactions using acoustic microstreaming

Expressão do gene correlacionar com o comportamento das células deve ser feito preferencialmente ao nível de uma única célula. No entanto, este não é facilmente alcançado porque a pequena quantidade de mRNA lábil presente em uma única célula (1-5% de 1-50pg RNA total, ou 0,01 2.5pg-mRNA, por célula 1) na maior parte degrada antes que possa ser transcrito reverso em uma cópia estável cDNA. Por exemplo, usando reagentes de laboratório padrão e hardware, apenas um pequeno número de genes podem ser avaliados qualitativamente por célula 2. Uma maneira de aumentar a eficiência do padrão reverso laboratório transcriptase (RT) reações (reagentes ou seja padrão em volumes microlitro) compreendendo uma única célula quantidades de mRNA seria mais rapidamente misturar os reagentes de modo que o mRNA pode ser convertido em cDNA antes que ele se degrada. No entanto, este não é trivial, pois em escalas microlitro fluxo de líquido é laminar, isto é, métodos atualmente disponíveis de misturar (isto é, agitação, vórtex e trituração) deixar de produzir movimento caótico suficiente para efetivamente misturar reagentes. Para resolver este problema, micro-escala de técnicas de mixagem tem que ser usado 3,4. Uma série de microfluídicos tecnologias baseadas em mistura, foram desenvolvidas com sucesso aumentar o rendimento de reação RT 5-8. No entanto, as tecnologias de microfluídica requerem hardware especializado que é relativamente caro e ainda não amplamente disponível. Uma solução mais barata, mais conveniente é desejável. O objetivo principal deste estudo é demonstrar como a aplicação de uma nova técnica &quot;micromixing&quot; a norma reações laboratório RT composto de uma única célula quantidades de mRNA aumenta significativamente sua produção de cDNA. Encontramos rendimentos cDNA aumentar em cerca de 1-10 vezes, o que permite: (1) um maior número de genes a ser analisados ​​por célula; (2) uma análise mais quantitativa da expressão do gene, e (3) melhor detecção de baixa abundância genes em células individuais. O micromixing é baseado em acústica microstreaming 12/09, um fenômeno onde as ondas sonoras propagando em torno de um pequeno obstáculo criar uma vazão média perto do obstáculo. Temos desenvolvido um dispositivo baseado em microstreaming acústica (&quot;micromisturador&quot;) com uma simplificação chave; microstreaming acústico pode ser alcançado em freqüências de áudio, assegurando o sistema tem uma interface líquido-ar com um pequeno raio de curvatura 13. O menisco de um volume microlitro de solução em um tubo fornece um raio de curvatura pequeno adequadamente. O uso de freqüências de áudio significa que o hardware pode ser barato e versátil 13, e ácidos nucléicos e outros reagentes bioquímicos não são danificadas, como eles podem estar com sonicators padrão de laboratório.

Watch this Scientific Journal Video about Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming at JoVE.com

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Descrevemos um novo método para aumentar a produtividade cDNA de uma única célula quantidades de mRNA em outra padrão de laboratório reações de transcrição reversa. A novidade reside na utilização de um micromisturador, que utiliza o fenômeno da microstreaming acústico, para misturar fluidos em escalas microlitro mais eficaz do que tremer, vórtex ou trituração.

Aumentar os rendimentos cDNA Single-cell quantidades de mRNA em Laboratório Padrão Reações transcriptase reversa usando Microstreaming Acústico

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

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Bioengineering

10.1K visualizações.  University of Melbourne. Descrevemos um novo método para aumentar a produtividade cDNA de uma única célula quantidades de mRNA em outra padrão de laboratório reações de transcrição reversa. A novidade reside na utilização de um micromisturador, que utiliza o fenômeno da microstreaming acústico, para misturar fluidos em escalas microlitro mais eficaz do que tremer, vórtex ou trituração.

Vídeo: Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Criando poros celulares transitórios membrana utilizando uma impressora jato de tinta padrão

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

Bioengenharia

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 ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Uma plataforma Microfluidic de ultra alta taxa de transferência para o sequenciamento do genoma de célula única

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

We describe a strategy for how to use RNA samples from unreferenced Pacific oyster specimens, and evaluate the genetic material by comparison with publicly available genome data to generate a virtually sequenced cDNA library.

Uma estratégia convergente para a geração de uma biblioteca de cDNA virtualmente seqüenciada de ostras pacíficos não referenciadas

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

Aumentando a Durabilidade de Culturas de Células Neurais Dissociadas Usando Matriz Coralina Biologicamente Ativa

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Esclarecimento Primário do Fluido de Cultura Celular Colhida cho usando um separador acústico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Standard Test Method ASTM D 7998-19 for the Cohesive Strength Development of Wood Adhesives

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the adhesive cure, and the effect of adhesive penetration on the wood interphase; thus, normal testing of a neat adhesive film is generally not useful. Most tests of wood adhesive bond strength are slow, laborious, can be strongly influenced by the wood and do not provide information on the kinetics of cure. Test method ASTM D 7998-19, however, can be used for fast evaluation of the strength of wood bonds. The use of a smooth, uniform, and strong wood surface, like maple face-veneer, and sufficient bonding pressure reduces the adhesion and wood strength effects on bond strength. This method has three main applications. The first is to provide consistent data on bond strength development. The second is to measure the dry and wet strengths of bonded lap shear samples. The third is to better understand the adhesive heat resistance by quickly evaluating thermal sensitivity and distinguishing between thermal softening and thermal degradation.

We present a procedure, ASTM D7998-19, for a rapid and more consistent evaluation of both dry and wet strength of adhesive bonds on wood. The method can also be used to provide information on strength development as a function of temperature and time or strength retention up to 250 &#176;C.

Método de Teste Padrão ASTM D 7998-19 para o Desenvolvimento de Resistência Coesiva de Adesivos de Madeira

The properties of cured wood adhesives are difficult to study because of the loss of water and other components to the wood, the influence of wood on the ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Microbolhas magnéticas, acústicas e óptica-tripla-responsivas para hipertermia magnética e terapia de câncer de combinação pothototérmica

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Medição potencial de ação óptica unicelular em cardiomiócitos derivados de células-tronco pluripotentes induzidos por humanos

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

This work presents a flexible protocol for utilizing fluorescently labeled elastomeric contractible surfaces (FLECS) Technology in microwell format for simplified, hands-off quantification of single-cell contractile forces based on visualized displacements of fluorescent protein micropatterns.

Análise simplificada e de alto rendimento da contractilidade unicelular usando Elastômeros Micropatterned

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, ...