Cette étude a été soutenue par la National Health and Medical Research Council de l&#39;Australie (projet de subvention non. 6.288.480) et le Scobie et Clare MacKinnon Trust.

1. Micromélange une réaction de RT <ol><li> Avant d&#39;effectuer une réaction de RT avec micromélange, à équilibrer le micromélangeur à la température désirée de la réaction de RT. <ol><li> Placez le dans un micromélangeur 37 ° C (ou la température recommandée par le fournisseur RT) incubateur pour au moins 1 heure avant le début de la réaction de RT. </li></ol></li><li> Mettre en place le mix RT en fonction de la transcriptase inverse du fournisseur (par exemple, MMTV-RT de Promega, Omniscript de Qiagen) des instructions, sauf à usage unique cellule du total des quantités d&#39;entrée ARN (par exemple 0.1-1pg/μl), au lieu de la g-ng quantités recommandées par les fournisseurs. <ol><li> Nous utilisons la norme stérile, sans nucléase 200 pl à parois minces tubes PCR. </li></ol></li><li> Position des tubes RT solidement dans le micromélangeur dès qu&#39;ils sont prêts pour le mixage. Le micromélangeur devrait rester à l&#39;intérieur de l&#39;incubateur 37 ° C pendant la durée de la réaction de RT. </li><li> Sélectionnez la fréquence appropriée et l&#39;amplitude de micromélange. Dans cet exemple nous utilisons 150Hz. </li><li> Allumez le micromélangeur le et laissez-le pendant toute la durée de la réaction de RT (60 minutes). </li><li> Allumez le micromélangeur off et de prendre les tubes RT une fois la réaction de RT est terminée. </li><li> Placer les tubes sur la glace RT et procéder comme normal avec d&#39;autres applications telles que Polymerase Chain Reaction quantitative (qPCR ou PCR en temps réel). </li></ol> 2. Les résultats représentatifs: L&#39;avantage de micromélange dans le contexte des réactions RT est une augmentation du rendement par rapport à l&#39;ADNc lorsque d&#39;autres méthodes de mélange (par exemple, secouer, ou vortex trituration) sont utilisés. Ceci peut être vu, par exemple, sur l&#39;analyse ultérieure de l&#39;ADNc en utilisant la polymérase quantitative Réaction chaîne (qPCR ou PCR en temps réel). La figure 2A illustre les effets de micromélange pour les 5 premières minutes (en bleu &quot;, micromixed5&quot;) ou les 60 minutes toute (rouge, &quot;micromixed60&quot;) par rapport à la trituration de la réaction de RT immédiatement avant l&#39;incubation RT (noir », la trituration&quot; ). Dans ce cas, le volume de la réaction précédente RT a été 25 pi et il contenait 2.5pg d&#39;ARN total. traces qPCR utilisant des amorces conçues pour détecter les ADNc NURR-1 représente l&#39;ARNm sont présentés ci-dessus, et les moyens ± SEM du nombre de cycles pour atteindre la fluorescence maximale de 50% en 3 répétitions de cette même expérience sont tracées ci-dessous. L&#39;astérisque indique une différence statistiquement significative (ANOVA à un facteur avec des comparaisons multiples de Tukey). Notez que pour le micromélange les 5 premières minutes de la réaction précédente RT produit une amélioration non significative au cours de trituration alors micromélange pour les 60 minutes toute la réaction précédente RT a produit une amélioration significative par rapport trituration, dont l&#39;ampleur était équivalente à environ 15 qPCR cycles, en moyenne. L&#39;avantage de micromélange peut également être vu dans la fonte analyse de la courbe des produits obtenus qPCR ultérieures. Dans l&#39;expérience illustrée dans la figure 2B, micromélange pour les 60 minutes toute la réaction précédente RT abouti à un signal qPCR cohérent dans des échantillons en double (figure 2BA, 2 traces rouges), tandis que micromélange pour les 5 premières minutes ou la trituration ne produit qPCR incompatibles des traces (figure 2BA, 2 bleus et 2 traces traces noires, respectivement). Les traces grises sont des contrôles négatifs («neg.&quot;) Dans lequel l&#39;ADNc a été écarté de la réaction PCR quantitative. Lorsque les produits qPCR de cette expérience ont été dénaturées par ramping leur température (ie analyse la courbe de fusion), il était évident que appropriée des produits de PCR (ie un amplicon qui était identique à celle obtenue lorsque les concentrations beaucoup plus élevées d&#39;ARNm ont été reverse transcrit et amplifié par PCR quantitative, données non présentées) étaient présents seulement suite aux réactions RT qui avait été micromixed pendant 60 minutes (figure 2Bb, 2 traces rouges). Tous les autres (c&#39;est à dire micromixed5, la trituration et des contrôles négatifs) générés produits alternatifs amplicon (dimères d&#39;amorces, par exemple). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Figure 1"/> Figure 1. Schéma du protocole d&#39;application de la norme de laboratoire micromélange réactions RT. Des descriptions plus détaillées du principe physique en cause, le micromélangeur et comment construire une micromélangeur en interne sont fournis dans les références 13,14 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Figure 2"/> Figure 2. Représentant de données qPCR illustrant les avantages de micromélange appliquée aux précédentes réactions RT comprenant une seule cellule quantités d&#39;ARNm dans les autres standards des réactions RT laboratoire. Traces qPCR Un exemple détecter l&#39;ARNm de l&#39;ADNc codant pour la représenter NURR-1 du gène. Les réactions précédentes RT contenue 2.5pg d&#39;ARN total dans un volume de 25 pi et ont été réalisées dans un incubateur à 37 ° C pendant 60 minutes. Réactions RT ont été mélangés par la trituration de la RT mélanger avant d&#39;incubation (traces noires », la trituration»), micromixed pendant les 5 premières minutes de la réaction de RT (traces bleues, &quot;micromixed5») ou micromixed pour les 60 minutes de toute la réaction de RT (traces rouges », micromixed60&quot; ). Moyens ± SEM du nombre de cycles nécessaires pour atteindre qPCR fluorescence maximale de 50% sont tracées ci-dessous pour n = 3 répétitions de cette expérience. L&#39;astérisque indique une différence significative (ANOVA à un facteur avec des comparaisons multiples de Tukey). B Une autre expérience qPCR examinant NURR-1 produit d&#39;ADNc, cette fois suite aux réactions RT contenant 25pg d&#39;ARN total dans un volume de 25 pi. traces qPCR (a) ainsi que la fonte analyse de la courbe des produits obtenus qPCR (b) sont indiqués pour les différentes conditions de mélange. Des contrôles négatifs dans lequel aucun ADNc a été inclus dans la réaction précédente RT sont également inclus (traces grises », neg.&quot;). Réactions RT avec des concentrations beaucoup plus élevées d&#39;ARNm ont également été réalisées et produites pics courbe de fusion (données non présentées) identiques à ceux de (b) pour micromixed60 (traces rouges) des échantillons.

<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>

Les auteurs n&#39;ont rien à révéler.

La méthode d&#39;application de la norme de laboratoire micromélange réactions RT décrites ici peuvent, bien sûr, impliquent l&#39;ARNm récoltés par n&#39;importe quelle méthode (par exemple des cellules de lyse, microdissection laser de capture). Il peut également comprendre toute les marques ou les types de réactifs RT, n&#39;importe quelle température (dans la tolérance des matériaux de l&#39;micromélangeur), et toute période de temps. Par exemple, nous avons observé l&#39;amélioration des rendements...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> L&#39;ARN total a été isolé de pression congelés préparés intensément pour adultes tranches mésencéphale de souris </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA Kit </td><td> Arcturus, CA, USA </td><td> KIT0204 </td><td> Le kit est désormais disponible auprès de Applied Biosystems </td></tr><tr><td> Sans ADN traitement à la DNase et réactifs de suppression </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Hexamère aléatoire amorces </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 jeu </td><td> Promega </td><td> U1240 </td><td></td></tr><tr><td> RNasine Inhibiteur de la ribonucléase </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Eau sans nucléase </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Vert PCR Master Mix </td><td> Applied Biosystem </td><td> 4309155 </td><td></td></tr><tr><td> Hprt avant (20mer): CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> Hprt inverse (20mer): TAT GTC CCC TGA CTG CGT AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 avant (23mer): CAG CCT CGA TTT CTT TCC CAA AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 inverse (23mer): GGT GAG GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 Spectrophotomètre. </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 a été acquise par QIAGEN en Juillet 2008 </td></tr></tbody></table>

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

L&#39;expression des gènes en corrélation avec le comportement des cellules est idéalement effectué au niveau unicellulaire. Cependant, ce n&#39;est pas facile à réaliser car la petite quantité d&#39;ARNm labiles présents dans une seule cellule (1-5% de 1-50pg ARN total, soit 0,01 2.5pg-ARNm, par cellule 1) dégrade la plupart du temps avant qu&#39;elle puisse être une transcription inverse dans une copie d&#39;ADNc stables. Par exemple, en utilisant des réactifs de laboratoire standard et matériel, seul un petit nombre de gènes peut être évaluée qualitativement par cellule 2. Une façon d&#39;augmenter l&#39;efficacité de la norme de laboratoire de la transcriptase inverse (RT) des réactions (réactifs standard dans des volumes microlitre) comprenant une seule cellule quantités d&#39;ARNm serait plus rapide mélanger les réactifs pour l&#39;ARNm peut être converti en ADNc avant qu&#39;il ne se dégrade. Toutefois, ce n&#39;est pas anodin car à l&#39;échelle du microlitre écoulement du liquide est laminaire, c&#39;est à dire les méthodes actuellement disponibles de mélange (c&#39;est à dire secouer, vortex et trituration) ne parviennent pas à produire suffisamment de mouvement chaotique efficacement mélanger les réactifs. Pour résoudre ce problème, micro-échelle des techniques de mélange doivent être utilisées 3,4. Un certain nombre de technologies basées sur microfluidiques mélange ont été développés avec succès d&#39;augmenter les rendements de réaction RT 5-8. Cependant, les technologies microfluidiques nécessitent du matériel spécialisé qui est relativement cher et n&#39;est pas encore largement disponibles. Une solution moins coûteuse, plus pratique est souhaitable. L&#39;objectif principal de cette étude est de démontrer comment l&#39;application d&#39;un roman &quot;micromélange&quot; technique de laboratoire standard réactions RT comprenant une seule cellule quantités d&#39;ARNm augmente considérablement leurs rendements d&#39;ADNc. Nous trouvons des rendements d&#39;ADNc augmenter d&#39;environ 10 à 100 fois, ce qui permet: (1) un plus grand nombre de gènes à analyser par cellule; (2) une analyse plus quantitative de l&#39;expression des gènes, et (3) une meilleure détection des gènes de faible abondance dans des cellules individuelles. Le micromélange est basé sur acoustiques microstreaming 9-12, un phénomène où les ondes sonores se propageant autour d&#39;un petit obstacle créer un débit moyen près de l&#39;obstacle. Nous avons développé une acoustique microstreaming basée sur l&#39;appareil («micromélangeur&quot;) avec une simplification clés; microstreaming acoustiques peuvent être obtenus à des fréquences audio en s&#39;assurant que le système a une interface liquide-air avec un petit rayon de courbure 13. Le ménisque d&#39;un volume microlitre de solution dans un tube fournit un rayon suffisamment petit de courbure. L&#39;utilisation des fréquences audio signifie que le matériel peut être peu coûteux et polyvalents 13, et des acides nucléiques et d&#39;autres réactifs biochimiques ne sont pas endommagés comme ils peuvent être avec sonicateurs de laboratoire standard.

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

Nous décrivons une nouvelle méthode pour augmenter le rendement d&#39;ADNc de cellule unique quantités d&#39;ARNm dans les réactions de laboratoire standard de transcription inverse contraire. La nouveauté réside dans l&#39;utilisation d&#39;un micromélangeur, qui utilise le phénomène de la microstreaming acoustique, de mélanger les liquides à l&#39;échelle du microlitre plus efficacement que secouer, vortex ou trituration.

L&#39;augmentation des rendements d&#39;ADNc de cellule unique quantités d&#39;ARNm dans les réactions de laboratoire standard de la transcriptase inverse en utilisant Microstreaming acoustique

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 ...

increasing-cdna-yields-from-single-cell-quantities-mrna-standard

Research

JoVE Journal

Bioengineering

10.1K vues.  University of Melbourne. Nous décrivons une nouvelle méthode pour augmenter le rendement d'ADNc de cellule unique quantités d'ARNm dans les réactions de laboratoire standard de transcription inverse contraire. La nouveauté réside dans l'utilisation d'un micromélangeur, qui utilise le phénomène de la microstreaming acoustique, de mélanger les liquides à l'échelle du microlitre plus efficacement que secouer, vortex ou trituration.

Vidéo: 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.

Création transitoires pores de la membrane cellulaire à l&#39;aide d&#39;une imprimante jet d&#39;encre standard

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

Recherche

Bio-ingénierie

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.

Une technique Label-free pour l&#39;imagerie spatio-temporelle de cellule unique sécrétions

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.

Une plate-forme de Microfluidic ultra haute capacité pour le séquençage du génome de cellules individuelles

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.

Une stratégie convergente pour la génération d'une bibliothèque d'ADNc pratiquement séquencée à partir d'huîtres du Pacifique non référencées

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.

Augmentation de la durabilité des cultures de cellules neurales dissociées à l’aide d’une matrice coralline biologiquement active

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.

Clarification primaire du fluide de culture cellulaire récolté par CHO à l’aide d’un séparateur acoustique

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.

Méthode d’essai normalisée ASTM D 7998-19 pour le développement de la résistance à la cohésion des adhésifs pour bois

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.

Microbulles magnétiques, acoustiques et optiques triple-réactives pour l’hyperthermie magnétique et la thérapie du cancer combinée pothototherme

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.

Mesure du potentiel d’action optique unicellulaire dans les cardiomyocytes dérivés de cellules souches pluripotentes induites par l’homme

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.

Analyse simplifiée et à haut débit de la contractilité unicellulaire à l’aide d’élastomères à micromotifs

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