Este estudio fue apoyado por el Sistema Nacional de Salud y el Consejo de Investigación Médica de Australia (proyecto de subvención no. 6.288.480) y el Scobie y Clare MacKinnon Trust.

1. Micromezcla una reacción RT <ol><li> Antes de realizar una reacción de RT con micromezcla, equilibrar la micromixer a la temperatura deseada de la reacción de RT. <ol><li> Coloque el micromixer dentro de 37 ° C (o la temperatura recomendada por el proveedor RT) incubadora de al menos 1 hora antes del inicio de la reacción de RT. </li></ol></li><li> Establecer la mezcla de RT de acuerdo con el proveedor de la transcriptasa inversa de (por ejemplo, MMTV-RT de Promega, Omniscript de Qiagen) instrucciones, excepto el uso de una sola célula cantidades de ARN total de entrada (por ejemplo, 0.1-1pg/μl), en lugar de la g-ng cantidades recomendadas por los proveedores. <ol><li> Utilizamos estándar estéril, libre de nucleasa 200μL tubos de pared delgada PCR. </li></ol></li><li> Posición RT tubos firmemente en la micromixer tan pronto como estén listos para mezclar. El micromixer debe permanecer dentro de la incubadora a 37 ° C durante la duración de la reacción de RT. </li><li> Seleccione la frecuencia adecuada y la amplitud de micromezcla. En este ejemplo vamos a usar 150Hz. </li><li> Cambiar el micromixer adelante y dejar actuar durante toda la duración de la reacción de RT (60 minutos). </li><li> Cambiar el micromixer fuera y tomar los tubos RT una vez que la reacción de RT se ha completado. </li><li> Coloque los tubos de RT en el hielo y continuar de forma normal con otras aplicaciones como la reacción en cadena de polimerasa cuantitativa (qPCR o PCR en tiempo real). </li></ol> 2. Los resultados representativos: El beneficio de micromezcla en el contexto de las reacciones de RT es un aumento en el rendimiento de cDNA en relación con los otros métodos de mezcla (por ejemplo, temblores, agitación o trituración) se utilizan. Esto se puede ver, por ejemplo, en el análisis posterior de los cDNA utilizando cuantitativa de reacción en cadena de la polimerasa (PCR cuantitativa o PCR en tiempo real). Figura 2A muestra los efectos de micromezcla durante los primeros 5 minutos (azul, &quot;micromixed5&quot;) o la totalidad de 60 minutos (de color rojo, &quot;micromixed60&quot;), en comparación con la trituración de la reacción de RT inmediatamente antes de la incubación RT (negro, &quot;trituración&quot; ). En este caso el volumen de la anterior reacción de RT se 25μL y contenía 2.5pg de ARN total. rastros qPCR utilizando los primers diseñados para detectar cDNA Nurr-1 representa ARNm se muestra arriba, y las medias ± SEM del número de ciclos para alcanzar el 50% máximo de fluorescencia en 3 repeticiones de este mismo experimento se representan a continuación. El asterisco indica una diferencia estadísticamente significativa (ANOVA de un factor con comparaciones múltiples de Tukey). Tenga en cuenta que micromezcla para los primeros 5 minutos de la anterior reacción RT producido una mejora no significativa en la trituración, mientras que para toda la micromezcla 60 minutos de la anterior reacción RT producido una mejora significativa en la trituración, la magnitud de lo que equivale a aproximadamente el 15 qPCR ciclos, en promedio. El beneficio de micromezcla también se puede ver en análisis de la curva de fusión de los productos obtenidos qPCR posteriores. En el experimento ilustrado en la Figura 2B, micromezcla para todo el 60 minutos de la anterior reacción RT dio lugar a una señal de qPCR consistente en duplicados de las muestras (Figura 2Ba, 2 rastros de color rojo), mientras que micromezcla durante los primeros 5 minutos o trituración sólo se produce qPCR inconsistente rastros (Figura 2Ba, dos azules y dos rastros rastros negro, respectivamente). Las huellas grises son los controles negativos (&quot;neg.&quot;) En el que cDNA fue dejado fuera de la reacción de qPCR. Cuando los productos qPCR de este experimento fueron desnaturalizados por la rampa de su temperatura (es decir, análisis de la curva de fusión), era evidente que los productos de PCR apropiada (es decir, una amplificación que era idéntica a la obtenida en concentraciones mucho más elevadas de ARNm se transcribe inversa y amplificado por PCR cuantitativa, datos no mostrados) sólo estaban presentes las siguientes reacciones de RT que se había micromixed durante 60 minutos (Figura 2Bb, 2 rastros de color rojo). Todos los demás (es decir, micromixed5, trituración y controles negativos) generado productos alternativos de amplificación (por ejemplo, dímeros de primers). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Figura 1"/> Figura 1. Diagrama de flujo del protocolo para la aplicación de micromezcla a métodos de laboratorio las reacciones de RT. Descripciones más detalladas de los principios físicos involucrados, micromixer y cómo construir un micromixer de la casa se ​​proporcionan en las referencias 13,14 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Figura 2"/> Figura 2. Representante qPCR datos que ilustran los beneficios de micromezcla se aplica a las reacciones de RT anterior que comprende una sola célula cantidades de ARNm de otra manera estándar de laboratorio las reacciones de RT. Un ejemplo de las huellas qPCR detección de ARNm cDNA que codifica para la representación de Nurr-1 gen. Lo anterior reacciones de RT contiene 2.5pg de ARN total en un volumen de 25μL y se llevaron a cabo en una incubadora a 37 ° C durante 60 minutos. RT reacciones fueron mezclados por trituración de la RT mezcla antes de la incubación (huellas negro &quot;trituración&quot;), micromixed durante los primeros 5 minutos de la reacción RT (huellas azules &quot;, micromixed5&quot;) o para toda la micromixed 60 minutos de la reacción RT (trazas rojas &quot;, micromixed60&quot; ). Media ± SEM del número de ciclos de PCR cuantitativa para llegar al 50% máximo de fluorescencia se representan a continuación para n = 3 repeticiones de este experimento. El asterisco indica una diferencia significativa (ANOVA de un factor con comparaciones múltiples de Tukey). B Otro experimento qPCR examinar Nurr-1 cDNA producto, esta vez después de las reacciones de RT contiene 25pg de ARN total en un volumen de 25μL. rastros qPCR (a), así como análisis de la curva de fusión de los productos obtenidos qPCR (b) se muestran las condiciones de mezcla diferentes. Los controles negativos en los que no cDNA fue incluido en la anterior reacción RT también se incluyen (los rastros grises &quot;, neg.&quot;). Reacciones de RT con concentraciones mucho más elevadas de ARNm se realizaron y produjeron picos de la curva de fusión (datos no mostrados) idénticas a las de (b) para micromixed60 (trazas rojas) muestras.

<ol>
	<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>

Los autores no tienen nada que revelar.

El método de aplicación de micromezcla a métodos de laboratorio las reacciones de RT descritos aquí pueden, por supuesto, implica ARNm cosechado a través de cualquier método (por ejemplo, células de lisis, láser microdisección de captura). También puede comprender cualquiera de las marcas o tipos de reactivos RT, cualquier temperatura (dentro de la tolerancia de los materiales de la micromixer), y cualquier período de tiempo. Por ejemplo, hemos observado que mejora los rendimientos de cDNA a partir de las rea...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> ARN total fue aislado de broche de congelados aguda preparado rebanadas del cerebro medio de ratones adultos </td><td></td><td></td><td></td></tr><tr><td> PicoPure ARN Kit de aislamiento </td><td> Arcturus, CA, EE.UU. </td><td> KIT0204 </td><td> El kit está disponible a partir de Applied Biosystems </td></tr><tr><td> ADN libre de DNasa tratamiento y eliminación de reactivos </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Cebadores aleatorios hexamer </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 conjunto </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasin Inhibidor de ribonucleasa </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Agua libre de nucleasa </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Green PCR Master Mix </td><td> Applied Biosystems </td><td> 4309155 </td><td></td></tr><tr><td> Hprt adelante (20mer): CTT TGC TGA GGA GCT CCT TT </td><td></td><td></td><td></td></tr><tr><td> Hprt inversa (20mer): TAT GTC CCC CGT TGA CTG EN </td><td></td><td></td><td></td></tr><tr><td> Nurr1 adelante (23mer): CAG CTC CGA TTT CTT TCC AAC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 inversa (23mer): GAG GGT GCT GTC CAT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 espectrofotómetro. </td><td> Thermo Scientific </td><td></td><td></td></tr><tr><td> Corbett Rotor Gene RG-6000 </td><td> Corbett Ciencias de la Vida </td><td></td><td> Corbett Ciencias de la Vida fue adquirida por QIAGEN en julio de 2008 </td></tr></tbody></table>

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

La correlación de la expresión génica con el comportamiento celular está muy bien hecho a nivel de una sola célula. Sin embargo, esto no es fácil de lograr debido a que la pequeña cantidad de ARNm lábil presente en una sola célula (1-5% de 1-50pg ARN total, o ARNm de 0,01 2.5pg, por celda 1) en su mayoría se degrada antes de que se transcriben de forma inversa en una copia de cDNA estable. Por ejemplo, utilizando reactivos de laboratorio y de hardware, sólo un pequeño número de genes pueden ser evaluadas cualitativamente por la celda 2. Una forma de aumentar la eficiencia de los métodos de laboratorio de la transcriptasa inversa (RT), las reacciones (es decir, los reactivos estándar en los volúmenes de microlitros) que comprende una sola célula cantidades de ARNm sería confundir más rápidamente los reactivos para el ARNm puede ser convertido a cDNA antes de que se degrada. Sin embargo, esto no es trivial, ya que a escalas microlitro de flujo de líquido es laminar, es decir, los métodos actualmente disponibles de la mezcla (es decir, temblores, agitación y trituración) no produce un movimiento caótico suficiente para mezclar con eficacia los reactivos. Para resolver este problema, micro-escala de técnicas de mezcla que se utilizará 3,4. Una serie de microfluidos tecnologías basadas en la mezcla, se han desarrollado con éxito aumentar el rendimiento de la reacción RT 8.5. Sin embargo, las tecnologías de microfluidos requieren hardware especializado que es relativamente caro y que aún no están ampliamente disponibles. Una solución más barata, más cómoda es deseable. El objetivo principal de este estudio es demostrar cómo la aplicación de una novela &quot;micromezcla&quot; técnica estándar de laboratorio las reacciones de RT que comprende una sola célula cantidades de ARNm aumenta significativamente el rendimiento de sus ADNc. Nos encontramos con los rendimientos de cDNA un aumento de aproximadamente 10 a 100 veces, lo que permite: (1) un mayor número de genes a ser analizados por la célula, (2) análisis cuantitativo de la expresión génica, y (3) una mejor detección de los genes de baja abundancia en las células individuales. El micromezcla se basa en acústico microcorrientes 12.09, un fenómeno por el cual las ondas de sonido alrededor de propagación de un pequeño obstáculo crear un flujo promedio cerca del obstáculo. Hemos desarrollado una acústica microcorrientes dispositivo basado en (&quot;micromixer&quot;), con una simplificación clave; microcorrientes acústica se puede conseguir en las frecuencias de audio, asegurando que el sistema tiene una interfase líquido-aire con un pequeño radio de curvatura 13. El menisco de un volumen de microlitro de solución en un tubo ofrece un radio de curvatura apropiado pequeños. El uso de las frecuencias de audio significa que el hardware puede ser barato y versátil 13, y los ácidos nucleicos y otros reactivos bioquímicos no son dañados como pueden ser con sonicadores estándar de laboratorio.

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

Se describe un nuevo método para aumentar el rendimiento de cDNA a partir de una sola célula cantidades de ARNm en las reacciones de laboratorio estándar de lo contrario la transcripción inversa. La novedad reside en el uso de un micromixer, que utiliza el fenómeno de las microcorrientes acústica, para mezclar líquidos a escalas microlitro con más eficacia que temblores, agitación o trituración.

Aumentar el rendimiento de cDNA a partir de una sola célula cantidades de ARNm en las reacciones de laboratorio estándar de la transcriptasa inversa con microcorrientes acústica

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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Research

JoVE Journal

Bioengineering

10.1K vistas.  University of Melbourne. Se describe un nuevo método para aumentar el rendimiento de cDNA a partir de una sola célula cantidades de ARNm en las reacciones de laboratorio estándar de lo contrario la transcripción inversa. La novedad reside en el uso de un micromixer, que utiliza el fenómeno de las microcorrientes acústica, para mezclar líquidos a escalas microlitro con más eficacia que temblores, agitación o trituración.

Video: 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.

Crear transitoria poros de la membrana de células utilizando una impresora de inyección de tinta estándar

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

Investigación

Bioingeniería

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.

Una técnica-Label libre para el Imaging espacio-temporal de las secreciones sola 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.

Una plataforma de ultra rendimiento microfluidos para la secuenciación del genoma de unicelular

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.

Una estrategia convergente para la generación de una biblioteca de ADNc virtualmente secuenciada a partir de ostras del Pacífico sin referencia

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.

Aumento de la durabilidad de cultivos de células neurales disociadas utilizando matriz coralina biológicamente activa

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.

Clarificación primaria del fluido de cultivo celular cosechado con CHO utilizando un 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 prueba estándar ASTM D 7998-19 para el desarrollo de la resistencia cohesiva de adhesivos para madera

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.

Microburbujas magnéticas, acústicas y ópticas de triple respuesta para la hipertermia magnética y la terapia combinada de cáncer potototérmico

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.

Medición del potencial de acción óptica unicelular en cardiomiocitos derivados de células madre pluripotentes inducidas 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.

Análisis simplificado y de alto rendimiento de la contractilidad de una sola célula utilizando elastómeros con micropatrones

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