Diese Studie wurde von der National Health and Medical Research Council of Australia (Projekt Grant No. 6288480) und der Scobie und Clare MacKinnon Trust unterstützt.

1. Mikrovermischung RT Reaction <ol><li> Vor der Durchführung einer RT-Reaktion mit Mikrovermischung, bringen die Mikromischer auf die gewünschte Temperatur der RT-Reaktion. <ol><li> Legen Sie die Mikromischer in einem 37 ° C (oder die Temperatur durch die RT Lieferant empfohlen) Inkubator für mindestens 1 Stunde vor Beginn der RT-Reaktion. </li></ol></li><li> Richten Sie die RT-Mix nach dem Reverse-Transkriptase-Lieferanten (zB MMTV-RT von Promega, Omniscript von Qiagen) Anweisungen, außer Nutzung Single-Cell-Mengen-Eingang Gesamt-RNA (z. B. 0.1-1pg/μl), anstelle der ng-pg Beträge, die Lieferanten zu empfehlen. <ol><li> Wir verwenden Standard-sterile, Nuklease-freies 200 ul dünnwandigen PCR-Gefäße. </li></ol></li><li> Position RT Rohre sicher in den Mikromischer, sobald sie bereit sind für das Mischen sind. Die Mikromischer sollte innerhalb der 37 ° C Inkubator für die Dauer der RT-Reaktion bleiben. </li><li> Wählen Sie die gewünschte Frequenz und Amplitude Mikrovermischung. In diesem Beispiel verwenden wir 150Hz. </li><li> Schalten Sie den Mikromischer auf und lassen Sie es für die gesamte Dauer der RT-Reaktion (60 Minuten). </li><li> Schalten Sie den Mikromischer aus und nehmen Sie die RT Rohre aus, wenn die RT-Reaktion abgeschlossen ist. </li><li> Legen Sie die RT Röhrchen auf Eis, und fahren wie gewohnt mit weiteren Anwendungen wie quantitative Polymerase-Kettenreaktion (qPCR oder real-time PCR). </li></ol> 2. Repräsentative Ergebnisse: Der Vorteil der Mikrovermischung im Rahmen der RT-Reaktionen ist eine Erhöhung der cDNA Ausbeute bezogen auf, wenn andere Mischverfahren (zB Zittern, Vortexen oder Verreibung) verwendet werden. Dies zeigt sich zum Beispiel bei der anschließenden Analyse der cDNA mittels quantitativer Polymerase-Kettenreaktion (qPCR oder real-time PCR) werden. Abbildung 2A zeigt die Auswirkungen der Mikrovermischung für die ersten 5 Minuten (blau, &quot;micromixed5&quot;) oder den gesamten 60 Minuten (rot, &quot;micromixed60&quot;), verglichen mit Verreibung der RT-Reaktion unmittelbar vor der RT Inkubation (schwarz, &quot;Verreiben&quot; ). In diesem Fall wird die Lautstärke des vorangegangenen RT-Reaktion wurde 25 &amp;mgr; l, und es enthielt 2.5pg von Gesamt-RNA. qPCR Spuren unter Verwendung der Primer entwickelt, um cDNA repräsentiert Nurr-1 mRNA erkennen sind oben gezeigt, und die Mittelwerte ± SEM der Anzahl der Zyklen bis 50% maximale Fluoreszenz in 3 Wiederholungen desselben Experiments zu erreichen sind nachfolgend dargestellt. Das Sternchen bedeutet einen statistisch signifikanten Unterschied (one-way ANOVA mit Tukey multiple Vergleiche). Beachten Sie, dass Mikrovermischung für die ersten 5 Minuten des vorangegangenen RT-Reaktion eine nicht-signifikante Verbesserung gegenüber Verreibung hergestellt, während Mikrovermischung für den gesamten 60 Minuten der vorhergehenden RT-Reaktion zu einer signifikanten Verbesserung gegenüber Verreibung, deren Größe entsprach etwa 15 qPCR Zyklen im Durchschnitt. Der Vorteil der Mikrovermischung kann auch in Schmelzkurvenanalyse der nachfolgenden qPCR erhaltenen Produkte gesehen werden. In dem Experiment in Abbildung 2B dargestellt, Mikrovermischung für den gesamten 60 Minuten der vorhergehenden RT-Reaktion führte zu einer konsequenten qPCR-Signal in zwei Proben (Abbildung 2Ba, 2 rote Spuren), während Mikrovermischung für die ersten 5 Minuten oder Verreibung nur produziert inkonsistent qPCR Spuren (Abbildung 2Ba, 2 blaue Spuren und 2 schwarzen Spuren, jeweils). Der graue Spuren sind negative Kontrollen (&quot;neg.&quot;), In der cDNA aus der qPCR-Reaktion wurde nach links. Wenn die qPCR Produkten aus diesem Experiment wurden denaturiert durch Rampen ihre Temperatur (dh Schmelzkurvenanalyse) war es offensichtlich, dass geeignete PCR-Produkte (dh ein Amplikon derjenigen identisch ist, erhält man, wenn viel höhere Konzentrationen des mRNA revers transkribiert wurden und durch qPCR amplifiziert wurde, Daten nicht gezeigt) waren nur folgende RT Reaktionen, die für 60 Minuten (Abbildung 2Bb, 2 rote Spuren) war micromixed. Alle anderen (dh micromixed5, Zerreiben und negative Kontrollen) generiert alternative Amplikon Produkte (zB Primer-Dimere). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Fließschema des Protokolls für die Anwendung Mikrovermischung Standard-Labor-RT-Reaktionen. Genauere Beschreibungen des physikalischen Prinzips beteiligt, die Mikromischer und wie eine in-house Mikromischer bauen in Verweise 13,14 zur Verfügung gestellt <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Representative qPCR-Daten, welche die Vorteile der Mikrovermischung zu vorangegangenen RT-Reaktionen mit Single-Cell-Mengen von mRNA in sonst üblichen Labor-RT-Reaktionen eingesetzt. Ein Beispiel qPCR Spuren erkennen cDNA repräsentiert mRNA codiert Nurr-1-Gen. Die vorstehenden RT Reaktionen enthielten 2.5pg von Gesamt-RNA in einem Volumen von 25 &amp;mgr; l und wurden in einem Brutschrank bei 37 ° C für 60 Minuten durchgeführt. RT-Reaktionen wurden durch Verreiben der R gemischtT-Mix vor der Inkubation (schwarz Spuren &quot;, Verreibung&quot;), während der ersten 5 Minuten der RT-Reaktion (blau Spuren &quot;, micromixed5&quot;) micromixed oder micromixed für den gesamten 60 Minuten der RT-Reaktion (rot Spuren &quot;, micromixed60&quot; ). Mittelwert ± SEM der Anzahl der qPCR-Zyklen erforderlich, um 50% maximale Fluoreszenz zu erreichen sind nachfolgend für n = 3 dargestellt Wiederholungen dieses Experiments. Asterisk zeigt einen signifikanten Unterschied (one-way ANOVA mit Tukey multiple Vergleiche). B Ein weiterer qPCR Experiment untersucht Nurr-1 cDNA-Produkt, diesmal nach RT-Reaktionen, die 25pg von Gesamt-RNA in einem Volumen von 25 &amp;mgr; l. qPCR Spuren (a) sowie Schmelzkurvenanalyse der qPCR Produkten erhalten (b) sind für die verschiedenen Misch-Bedingungen gezeigt. Negative Kontrollen, in denen keine cDNA in den vorangegangenen RT-Reaktion enthalten war sind ebenfalls enthalten (graue Spuren &quot;, neg.&quot;). RT-Reaktionen mit sehr viel höheren Konzentrationen von mRNA wurden ebenfalls durchgeführt und produziert Schmelzkurve Peaks (Daten nicht gezeigt) identisch mit denen in (b) für micromixed60 (rote Kurven) Proben.

<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>

Die Autoren haben nichts zu offenbaren.

Die Methode der Anwendung von Mikrovermischung Standard-Labor-RT hier beschriebenen Reaktionen kann natürlich beinhalten mRNA geerntet über einen beliebigen Methode (zB Zell-Lyse-, Laser-Mikrodissektion). Es kann auch eine beliebige Marken oder Typen von RT-Reagenzien, bei jeder Temperatur (innerhalb der Toleranz von den Materialien der Mikromischer) und einen beliebigen Zeitraum. Zum Beispiel haben wir eine verbesserte cDNA Erträge aus RT-Reaktionen mit random Hexamer oder Oligo-dT-Primer beobachtet. Es könnte auch...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> Gesamt-RNA wurde aus schockgefroren akut vorbereitet erwachsenen Maus Mittelhirn Scheiben isoliert </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA Isolation Kit </td><td> Arcturus, CA, USA </td><td> KIT0204 </td><td> Das Kit ist ab sofort bei Applied Biosystems </td></tr><tr><td> DNA-freie DNase Behandlung und Beseitigung Reagenzien </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Zufällige Hexamer Primer </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 Ribonuclease Inhibitor </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Nuklease-freies Wasser </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 forward (20mer): CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> HPRT reverse (20mer): TAT GTC CCC CGT TGA CTG AT </td><td></td><td></td><td></td></tr><tr><td> Nurr1 forward (23mer): CAG CTC CGA TTT CTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 reverse (23mer): GGT GAG GTC CAT GCT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 Spektralphotometer. </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 wurde von QIAGEN im Juli 2008 erworben </td></tr></tbody></table>

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

Die Korrelation der Genexpression mit Zellverhalten ist ideal an den Single-Cell-Ebene getan. Dies ist jedoch nicht leicht zu erreichen, weil die geringe Menge an labile mRNA in einer einzigen Zelle (1-5% der 1-50pg Gesamt-RNA oder 0,01-2.5pg mRNA, pro Zelle 1) meist abbaut, bevor sie transkribiert werden rückgängig machen kann in eine stabile cDNA-Kopie. Zum Beispiel unter Verwendung von Standard-Labor Reagenzien und Hardware können nur eine kleine Anzahl von Genen qualitativ pro Zelle 2 bewertet. Eine Möglichkeit, die Effizienz der Standard-Labor-Reverse-Transkriptase (RT)-Reaktionen (zB Standard-Reagenzien in mikrolitergroßen) mit Single-Cell-Mengen von mRNA zu erhöhen wäre, schneller zu mischen Reagenzien, so dass die mRNA in cDNA umgewandelt werden können, bevor es abgebaut wird. Dies ist jedoch nicht trivial, weil bei Mikroliter Flüssigkeit Skalen Strömung laminar, dh derzeit verfügbaren Methoden der Durchmischung (dh Zittern, Vortexen und Verreibung) nicht genügend chaotische Bewegung effektiv Reagenzien zu produzieren. Um dieses Problem zu lösen, haben Mikro-Maßstab Mischtechniken verwendet 3,4 sein. Eine Reihe von mikrofluidischen-basierte Misch-Technologien entwickelt worden, die erfolgreich erhöhen RT Reaktionsausbeuten 5-8. Allerdings erfordern Mikrofluidik-Technologien spezialisierte Hardware, die relativ teuer und noch nicht allgemein verfügbar ist. Eine billigere, bequemere Lösung ist wünschenswert. Das Hauptziel dieser Studie ist es zu demonstrieren, wie Einsatz eines neuartigen &quot;Mikrovermischung&quot; Technik, um Standard-Labor-RT-Reaktionen mit Single-Cell-Mengen von mRNA signifikant erhöht ihre cDNA ergibt. Wir finden cDNA Erträge um etwa 10 bis 100-fache, die ermöglicht zu erhöhen: (1) eine größere Anzahl von Genen pro Zelle analysiert werden, (2) mehr quantitative Analyse der Genexpression, und (3) eine bessere Erkennung von Low-Fülle Gene in einzelnen Zellen. Die Mikrovermischung auf akustische Microstreaming 9-12, ein Phänomen, wo die Schallwellen ausbreiten um ein kleines Hindernis zu schaffen eine mittlere Strömung in der Nähe des Hindernisses auf. Wir haben eine akustische Microstreaming-basierten Gerät (&quot;Mikromischer&quot;) mit einer Schlüssellänge Vereinfachung entwickelt, akustische Microstreaming kann bei Audio-Frequenzen, indem sichergestellt wird das System über einen Flüssigkeits-Luft-Schnittstelle mit einem kleinen Krümmungsradius 13 erreicht werden. Der Meniskus einer Mikroliter Volumen der Lösung in einer Röhre bietet eine entsprechend kleinen Krümmungsradius. Die Verwendung von Audio-Frequenzen bedeutet, dass die Hardware kann kostengünstig und vielseitig 13 und Nukleinsäuren und anderen biochemischen Reagenzien dürfen nicht beschädigt sein, wie sie mit Standard-Labor sonicators werden kann.

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

Wir beschreiben ein neuartiges Verfahren zur Erhöhung der cDNA-Ausbeute von Single-Cell-Mengen von mRNA in sonst üblichen Labor reverse Transkription Reaktionen. Die Neuheit besteht in der Verwendung von einem Mikromischer, die das Phänomen der akustischen Microstreaming nutzt, um Flüssigkeiten bei Mikroliter Skalen effektiver Mix als Schütteln, Vortexen oder Verreibung.

Steigende cDNA Erträge aus Einzel-Zell-Mengen von mRNA in Standard Laboratory Reverse-Transkriptase-Reaktionen mit Acoustic Microstreaming

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 Aufrufe.  University of Melbourne. Wir beschreiben ein neuartiges Verfahren zur Erhöhung der cDNA-Ausbeute von Single-Cell-Mengen von mRNA in sonst üblichen Labor reverse Transkription Reaktionen. Die Neuheit besteht in der Verwendung von einem Mikromischer, die das Phänomen der akustischen Microstreaming nutzt, um Flüssigkeiten bei Mikroliter Skalen effektiver Mix als Schütteln, Vortexen oder Verreibung.

Serie: 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.

Erstellen Transient Zelle Membranporen Mit einem Standard-Inkjet-Drucker

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

Forschung

Biotechnik

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.

Ein Markierungsfreie Technik für die Raum-zeitliche Imaging von Single Cell Sekrete

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.

Eine höchste Durchsatz mikrofluidischen Plattform für einzellige Genom-Sequenzierung

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.

Eine konvergierende Strategie für die Generierung einer virtually Sequenced cDNA Library aus unreferenzierten pazifischen Austern

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.

Erhöhung der Haltbarkeit dissoziierter neuronaler Zellkulturen unter Verwendung biologisch aktiver Korallenmatrix

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.

Primärklärung der CHO geernteten Zellkulturflüssigkeit mittels eines akustischen Separators

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.

Standardprüfverfahren ASTM D 7998-19 zur kohäsiven Festigkeitsentwicklung von Holzklebstoffen

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.

Magnetische, akustische und optische dreifach ansprechende Mikroblasen für magnetische Hyperthermie und pothotothermische Kombinationskrebstherapie

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.

Einzelzellige optische Aktionspotentialmessung in human induzierten pluripotenten Stammzell-abgeleiteten Kardiomyozyten

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.

Vereinfachte Hochdurchsatzanalyse der Einzelzellkontraktilität mit mikrostrukturierten Elastomeren

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