Questo studio è stato sostenuto dal National Health and Medical Research Council of Australia (progetto non concede. 6.288.480) e il Scobie e Chiara MacKinnon Trust.

1. Micromixing una reazione RT <ol><li> Prima di eseguire una reazione RT con micromixing, equilibrare la micromixer alla temperatura desiderata della reazione RT. <ol><li> Posizionare il micromixer all&#39;interno di un 37 ° C (o la temperatura consigliata dal fornitore RT) incubatore per almeno 1 ora prima dell&#39;inizio della reazione RT. </li></ol></li><li> Impostare il mix RT secondo il fornitore di trascrittasi inversa (ad esempio MMTV-RT da Promega, Omniscript da Qiagen) le istruzioni, tranne l&#39;uso di singole cellule quantità di RNA totale di ingresso (per esempio 0.1-1pg/μl), invece del-ng mg quantità raccomandate dai fornitori. <ol><li> Noi uso standard sterile, priva di nucleasi 200μL tubi con pareti sottili PCR. </li></ol></li><li> Posizione tubi RT saldamente il micromixer non appena sono pronti per la miscelazione. Il micromixer deve rimanere all&#39;interno della 37 ° C incubatore per tutta la durata della reazione RT. </li><li> Selezionare la corretta frequenza e l&#39;ampiezza di micromixing. In questo esempio si usa 150Hz. </li><li> Accendere il micromixer su e lasciarlo acceso per tutta la durata della reazione RT (60 minuti). </li><li> Accendere il micromixer fuori e prendere i tubi RT una volta la reazione di RT è completa. </li><li> Posizionare i tubi RT su ghiaccio e procedere come di consueto con ulteriori applicazioni quali quantitativa reazione a catena della polimerasi (qPCR o real-time PCR). </li></ol> 2. Rappresentante dei risultati: Il vantaggio di micromixing nel contesto di reazioni RT è un aumento della resa del DNA rispetto a quando altri metodi di miscelazione (es. agitazione, vortex o triturazione) sono utilizzati. Questo può essere visto, per esempio, su successiva analisi del cDNA Reazione a catena della polimerasi utilizzando quantitativa (qPCR o real-time PCR). Figura 2A illustra gli effetti di micromixing per i primi 5 minuti (blu, &quot;micromixed5&quot;) o l&#39;intero 60 minuti (rosso, &quot;micromixed60&quot;) rispetto a triturazione della reazione RT immediatamente prima della incubazione RT (nero, &quot;triturazione&quot; ). In questo caso il volume della reazione precedente RT era 25μL e conteneva 2.5pg di RNA totale. tracce qPCR utilizzando primers progettati per rilevare cDNA che rappresentano Nurr-1 mRNA sono indicati sopra, e la media ± SEM del numero di cicli per raggiungere fluorescenza massimo del 50% in 3 ripetizioni di questo stesso esperimento sono tracciate di seguito. L&#39;asterisco indica una differenza statisticamente significativa (ANOVA a una via con Tukey confronti multipli). Si noti che micromixing per i primi 5 minuti della reazione precedente RT ha prodotto una non significativa miglioramento rispetto triturazione micromixing mentre per l&#39;intero 60 minuti di reazione RT precedente ha prodotto un miglioramento significativo rispetto triturazione, la cui entità è stata pari a circa 15 qPCR cicli, in media. Il vantaggio di micromixing può essere visto anche in curva di fusione analisi dei prodotti qPCR successive ottenuti. Nell&#39;esperimento illustrato nella figura 2B, micromixing per l&#39;intero 60 minuti di reazione precedente RT portato ad un segnale qPCR consistente in campioni di duplicato (Figura 2BA, 2 tracce di rosso), mentre micromixing per i primi 5 minuti o triturazione prodotto solo qPCR incoerente tracce (Figura 2BA, 2 tracce blu e 2 tracce nere, rispettivamente). Le tracce grigio sono controlli negativi (&quot;neg.&quot;) In cui cDNA è stato lasciato fuori della reazione qPCR. Quando i prodotti qPCR da questo esperimento sono state denaturate dal rampa loro temperatura (analisi di fusione cioè curva) era evidente che determinati prodotti PCR (cioè un amplicone che era identica a quella ottenuta quando le concentrazioni molto più elevato di mRNA sono stati trascrizione inversa e amplificata da qPCR, dati non riportati) erano presenti solo le seguenti reazioni RT che erano stati micromixed per 60 minuti (Figura 2Bb, 2 tracce di rosso). Tutti gli altri (cioè micromixed5, triturazione e controlli negativi) di produzione di beni amplicone alternative (ad esempio dimeri primer). <img src="/files/ftp_upload/3144/3144fig1.jpg" alt="Figura 1"/> Figura 1. Diagramma di flusso del protocollo per l&#39;applicazione di standard di laboratorio micromixing reazioni RT. Descrizioni più dettagliate del principio fisico coinvolto, la micromixer e come costruire un in-house micromixer sono forniti riferimenti 13,14 <img src="/files/ftp_upload/3144/3144fig2.jpg" alt="Figura 2"/> Figura 2. Rappresentante dati qPCR illustrando i vantaggi della micromixing applicata al precedente reazioni RT composto da singole cellule quantità di mRNA in altro modo standard di laboratorio reazioni RT. Un esempio tracce qPCR rilevazione del DNA mRNA rappresenta la codifica Nurr-1 gene. Le reazioni precedente RT contenute 2.5pg di RNA totale in un volume di 25μL e sono stati eseguiti in un incubatore a 37 ° C per 60 minuti. Reazioni RT sono state remixate da triturazione del RT mix prima di incubazione (tracce nere, &quot;triturazione&quot;), micromixed durante i primi 5 minuti della reazione RT (tracce blu, &quot;micromixed5&quot;) o micromixed per l&#39;intero 60 minuti di reazione RT (tracce rosse, &quot;micromixed60&quot; ). Media ± SEM del numero di cicli necessari per raggiungere qPCR fluorescenza massimo del 50% sono riportati qui di seguito per n = 3 ripetizioni di questo esperimento. Asterisco indica una differenza significativa (ANOVA a una via con Tukey confronti multipli). B Un altro esperimento qPCR esaminando Nurr-1 prodotto cDNA, questa volta a seguito di reazioni RT contenente 25pg di RNA totale in un volume di 25μL. tracce qPCR (a), così come l&#39;analisi della curva di fusione dei prodotti qPCR ottenuti (b) sono indicati per le diverse condizioni di miscelazione. Controlli negativi in ​​cui è stato inserito nessun cDNA nella reazione precedente RT sono inclusi anche (tracce grigie, &quot;neg.&quot;). Reazioni RT con concentrazioni molto più elevate di mRNA sono stati eseguiti e prodotti picchi curva di fusione (dati non riportati) identici a quelli (b) per micromixed60 (tracce rosse) campioni.

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

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Il metodo di applicazione della micromixing a standard di laboratorio reazioni RT qui descritto può, ovviamente, coinvolgere mRNA raccolte attraverso qualsiasi mezzo (ad esempio la lisi cellulare, microdissezione laser cattura). Si può altresì comprendere qualsiasi marca o tipi di reagenti RT, qualsiasi temperatura (entro la tolleranza dei materiali della micromixer), e qualsiasi periodo di tempo. Per esempio, abbiamo osservato una migliore resa del DNA da reazioni di RT che comprende casuale esamero o oligo-dT prime...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> RNA totale è stato isolato da scatto fette surgelati del mouse acutamente preparato adulti mesencefalo </td><td></td><td></td><td></td></tr><tr><td> PicoPure RNA Isolation Kit </td><td> Arturo, CA, USA </td><td> KIT0204 </td><td> Il kit è ora disponibile da Applied Biosystems </td></tr><tr><td> DNA-free DNasi trattamento e reagenti di rimozione </td><td> Ambion </td><td> AM1906M </td><td></td></tr><tr><td> Casuale esamero 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 Inibitore di ribonucleasi </td><td> Promega </td><td> N2111 </td><td></td></tr><tr><td> Acqua priva di nucleasi </td><td> Promega </td><td> P1193 </td><td></td></tr><tr><td> SYBR Verde PCR Master Mix </td><td> Applied Biosystems </td><td> 4309155 </td><td></td></tr><tr><td> HPRT in avanti (20mer): CTT TGC TGA CCT GCT GGA TT </td><td></td><td></td><td></td></tr><tr><td> Hprt inversa (20mer): TAT GTC CCC CGT TGA CTG A </td><td></td><td></td><td></td></tr><tr><td> Nurr1 avanti (23mer): CAG CTC CGA CTT TTT AAC TCC AG </td><td></td><td></td><td></td></tr><tr><td> Nurr1 inversa (23mer): GGT GAG GTC CGT CAT AAA CTT GA </td><td></td><td></td><td></td></tr><tr><td> NanoDrop 1000 Spettrofotometro. </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 è stata acquisita da QIAGEN nel luglio 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;espressione genica correlazione con il comportamento delle cellule è idealmente fatto a livello di singola cellula. Tuttavia, questo non è facilmente ottenibile, perché la piccola quantità di mRNA labili presenti in una singola cella (1-5% di 1-50pg RNA totale, o 0,01 2.5pg mRNA, per cella 1) degrada soprattutto prima di poter essere trascrizione inversa in una copia stabile cDNA. Ad esempio, utilizzando reagenti di laboratorio e hardware standard, solo un piccolo numero di geni possono essere qualitativamente valutati per cella 2. Un modo per aumentare l&#39;efficienza dello standard di laboratorio della trascrittasi inversa (RT) reazioni (reagenti standard, cioè in volumi microlitri) comprendente cella singola quantità di mRNA potrebbe essere quella di mescolare più rapidamente i reagenti in modo che il mRNA può essere convertito in cDNA prima che degrada. Tuttavia questo non è banale perché a scale microlitro flusso del liquido è laminare, cioè i metodi attualmente disponibili di miscelazione (es. agitazione, vortex e triturazione) non riescono a produrre sufficiente moto caotico di mescolare in modo efficace i reagenti. Per risolvere questo problema, le tecniche di miscelazione micro-scala devono essere utilizzati 3,4. Un certo numero di tecnologie di miscelazione microfluidica a base sono state sviluppate con successo che aumentare le rese di reazione RT 5-8. Tuttavia, le tecnologie microfluidica richiedono hardware specializzato che è relativamente costoso e non ancora ampiamente disponibili. Una soluzione più economica, più conveniente è auspicabile. L&#39;obiettivo principale di questo studio è di dimostrare come l&#39;applicazione di una nuova tecnica &quot;micromixing&quot; per standard di laboratorio reazioni RT composto da singole cellule quantità di mRNA aumenta significativamente il loro rendimento cDNA. Troviamo rendimenti cDNA aumentare di circa 10 100 volte, che consente: (1) maggior numero di geni da analizzare per cella, (2) un&#39;analisi più quantitativa di espressione genica, e (3) rilevazione migliore di bassa abbondanza geni in singole cellule. Il micromixing si basa su acustica microstreaming 9-12, un fenomeno in cui le onde sonore di propagazione intorno ad un piccolo ostacolo creare un flusso medio vicino l&#39;ostacolo. Abbiamo sviluppato un acustico microstreaming dispositivo basato su (&quot;micromixer&quot;) con una semplificazione chiave; microstreaming acustico può essere ottenuto a frequenze audio, garantendo che il sistema è un liquido-aria con un piccolo raggio di curvatura 13. Il menisco di un volume microlitri di soluzione in un tubo fornisce un adeguato raggio di curvatura piccolo. L&#39;uso delle frequenze audio significa che l&#39;hardware può essere poco costoso e versatile 13, e gli acidi nucleici e di altri reagenti biochimici non sono danneggiati come possono essere con sonicatori standard di 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

Descriviamo un nuovo metodo per aumentare la resa cDNA da singole cellule quantità di mRNA nelle reazioni inverso altrimenti standard di laboratorio trascrizione. La novità risiede nell&#39;utilizzo di un micromixer, che utilizza il fenomeno della microstreaming acustico, di mescolare i fluidi a scale microlitro più efficacemente di agitazione, vortex o triturazione.

Aumentare le rese cDNA da singola cellula quantità di mRNA nelle reazioni standard di laboratorio della trascrittasi inversa utilizzando Microstreaming acustico

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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10.1K Views.  University of Melbourne. Descriviamo un nuovo metodo per aumentare la resa cDNA da singole cellule quantità di mRNA nelle reazioni inverso altrimenti standard di laboratorio trascrizione. La novità risiede nell'utilizzo di un micromixer, che utilizza il fenomeno della microstreaming acustico, di mescolare i fluidi a scale microlitro più efficacemente di agitazione, vortex o triturazione.

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.

Creazione transitori pori della membrana cellulare utilizzando una stampante a getto d&#39;inchiostro standard

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

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Bioingegneria

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 tecnica senza etichetta per l&#39;Imaging spazio-temporale delle secrezioni monocellulari

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 piattaforma di Microfluidic ad altissima velocità di trasmissione per il sequenziamento del genoma di cella singola

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 strategia convergente per la generazione di una libreria cDNA virtualmente in sequenza da ostriche del Pacifico senza riferimenti

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.

Aumentare la durabilità delle colture di cellule neurali dissociate utilizzando la matrice corallina biologicamente attiva

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.

Chiarificazione primaria del fluido di coltura cellulare raccolto da CHO utilizzando un separatore acustico

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.

Metodo di prova standard ASTM D 7998-19 per lo sviluppo della resistenza coesiva degli adesivi per legno

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.

Microbolle magnetiche, acustiche e ottiche a tripla risposta per ipertermia magnetica e terapia del cancro combinata pototermica

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.

Misurazione del potenziale d'azione ottico a singola cellula in cardiomiociti derivati da cellule staminali pluripotenti indotte umane

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.

Analisi semplificata e ad alta produttività della contrattilità a cella singola utilizzando elastomeri micropatterati

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