Die Autoren bedanken sich bei den Natural Sciences and Engineering Research Council of Canada, Canada Research Chairs Programm, Canadian Institutes of Health Research und Ontario Ministerium für Forschung und Innovation durch ihre frühe Forscher Award Programm für die finanzielle Unterstützung bedanken.

1. Photolithographie für Master Mold Fabrication <ol><li> Bathe eine 3 Zoll Siliziumwafer in Aceton 30 sec und dann mit Methanol für 30 sek. Spülen Sie mit dH 2 0 Wasser für 5 min. </li><li> Trocknen Sie die Oberfläche des Wafers mit einem N2 Blaspistole. Erhitzen des Wafers auf einer heißen Platte bei 140 ° C für 2 min. </li><li> Plasma die Oxidation der Oberfläche des Silizium-Wafers (1 min, 50 W). </li><li> Spin-Mantel der Oberfläche des Wafers mit 3 ml SU-8 Fotolack 100 (40 sec; 1.750 rpm). </li><li> Pre-Bake der beschichtete Wafer auf einer Heizplatte bei 65 ° C für 10 min, dann die Rampe die Temperatur bis zu 95 ° C über 2 min. Halten Sie diese Einstellung für eine zusätzliche 1 Stunde. </li><li> Richten Sie ein Photomaske mit dem gewünschten Kanal Design. Freilegen zu 550-600 mJ / cm 2 UV-Licht (350-400 nm) zu widerstehen. Photomasks können in AutoCAD entworfen und gedruckt auf einer Transparenz mit hoher Druckauflösung. </li><li> Nachhärten des Wafers auf einer heißen Platte bei 65 ° C für 1 min und 95 ° C für 10 min, Rampen ter Temperatur wie zuvor. </li><li> Tauchen Sie den Wafer in SU-8-Entwickler-Lösung für 10-15 min. Überprüfen Sie für die Fertigstellung der Entwicklung durch Spülen mit Isopropanol. Wenn ein weißer Niederschlag erscheint, weiter zu entwickeln. Die Master-Form ist in 2A gezeigt. </li></ol> 2. Soft-Lithographie für Microchannel Fabrication <ol><li> Mischen 35 ml Polydimethylsiloxan (PDMS) Elastomerbasis mit 3,5 ml PDMS Härtungsmittel. </li><li> Setzen Sie den Master hergestellt Form (Muster nach oben) und eine leere Siliziumwafer in Petrischalen mit Alufolie ausgekleidet. </li><li> Gießen Sie 20 ml PDMS Prepolymer in die Urform Schale und 15 ml in das zweite Gericht. Beseitigen Sie Lufteinschlüsse unter den Wafern durch leichten Druck auf sie mit einem Einweg-Holz-Applikator. </li><li> Titelbild beide Gerichte und beiseite für einen Tag, um zu heilen. Alternativ zur schnelleren Aushärtung entfernen Luftblasen aus dem PDMS mit einem Vakuumentgaseranlage und verlassen dann die Gerichte auf einer Heizplatte bei 80 ° C für 2hr. </li><li> Entfernen Sie die Folie und schälen die PDMS von den Wafern. </li><li> Verwenden Sie die Harris Uni-Core (2,5 mm), um Flüssigkeit Access Ports an beiden Enden des Kanals zu stanzen. Schneiden Sie den Kanal und leere PDMS in ähnlich große Streifen schneiden. </li><li> Legen Sie den Kanal, den Rohling PDMS-Streifen und einen Glasträger (75 × 25 mm 2) in ein Plasma Oxidationsmittel, wahrscheinlich in einem Reinraum befindet. Expose zum Sauerstoff-Plasma für 40 sec bei 40 W Leistung. </li><li> Haften die Kanalstück und Objektträger auf gegenüberliegenden Seiten des Rohlings Streifen. Beiseite für 2 Stunden, um die Bindung zu vervollständigen. </li><li> Legen Sie die Baugruppe auf einer Heizplatte bei 120 ° C. Befestigen Kunststoffschlauch (Innendurchmesser 1/32 &quot;, Außendurchmesser 3/32&quot;), die jeweils mindestens 6 mm lang, um die ausgestanzten Behältern mit PDMS Prepolymer. Bringen Sie eine fluidische Kunststoff-Stecker auf eine oder beide Röhren Spritze Befestigung ermöglichen, oder verwenden Sie handelsübliche Armaturen. </li><li> Lassen Sie das PDMS Sicherung der Schlauch zu heilen. Legen Sie 3 &quot;Längen von 22-Gauge-isoliertem Kupferdraht in each Reservoir, zwischen dem Eingang und dem Rohr-Kanal und sicher mit PDMS Prepolymer. Das fertige Produkt wird in 2B gezeigt. </li></ol> 3. Electrotaxis Experiment <ol><li> Legen Sie die Mikrokanalstruktur auf der Bühne (vorzugsweise XY-bewegliche) eines Mikroskops mit einer fest montierten Kamera mit einem Monitor verbunden (Abbildung 1). </li><li> Schließen Sie das Netzteil oder Ausgang des Verstärkers Drähte an der Mikrokanalstruktur der Elektroden. Eine einfache Gleichstromversorgung nicht ausreichend, wenn nur ein DC-Signal gewünscht wird, sondern ein Verstärker, der mit einem Funktionsgenerator ermöglicht Einsatz von gepulsten DC-und AC-Signale als auch. </li><li> Bringen Sie die Mikrokanalstruktur den Ausgang Rohr zu einer Einwegspritze. Tauchen der Mund des Einlaßrohres in M9 physiologischen Puffer und sanft streben Flüssigkeit in dem Kanal durch Anlegen eines Unterdrucks im Inneren der Spritze (entweder manuell oder unter Verwendung einer Spritzenpumpe). Wenn die Ein-und Auslass Rohre sowohl mit M9 gefüllt, ziehen Sie die Spritze from die Röhre. Wert, den beide Rohre auf die gleiche Höhe hydrostatisch angetriebene Strömung zu verhindern. </li><li> Anwenden einer Gleichspannung an den Kanal und zu gewährleisten, dass der Widerstand (R = V / I) beträgt etwa 0,6 MOhm (für eine 50 mm lang, 0,3 mm breit und 0,1 mm tief ~ Mikrokanal). </li><li> Wenn mit dem Kanal die Integrität erfüllt, befolgen Sie die obigen Schritte, um Würmer aus einer verdünnten Suspension laden in den Kanal. </li><li> Trennen Sie die Spritze und hydrostatisch manipulieren die durch Einstellen der Röhrchen relative Höhe. Verwenden Sie diese Methode, um einen Wurm in der Mitte des Kanals zu platzieren und dann legen die beiden Rohre flach auf der gleichen Höhe. </li><li> Stellen Sie die Stromversorgung auf die entsprechende Spannung: 4-12 V / cm für L3 Stadium Tiere, 4-10 V / cm für L4s und 2-4 V / cm für junge Erwachsene. Aktivieren Sie das elektrische Signal und damit 1 min von Vorbelichtung für die Schnecke auf dem Gebiet akklimatisieren. Der Wurm sollte beginnen, sich in Richtung der Kathode. Wenn die Minute vergangen ist, verwenden Sie die Kamera, um die Aufnahme zu starten. </li><li> Für AC-und Impuls-d DC Experimente, die maximale Reaktion elektrische Feld kann von oben und von Frequenz und Tastverhältnis des Signals angenommen werden kann wie gewünscht moduliert 7, 8. </li><li> Als Experiment beendet ist, entfernen Sie alle Flüssigkeit (und Würmer) aus dem Kanal, spülen Sie es mit dH 2 0, und lassen Sie das Gerät auf einer Heizplatte bei 125 ° C zu trocknen. </li><li> Auszug Bewegungsapparates Daten von aufgenommenen Videos manuell mit NIH ImageJ ( <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) oder benutzerdefinierte MATLAB-basierten Wurm-Tracking-Software. </li></ol>

<ol>
	<li>Kuwahara, T., Koyama, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familial+Parkinson+mutant+&#945;-synuclein+causes+dopamine+neuron+dysfunction+in+transgenic+Caenorhabditis+elegans.">Familial Parkinson mutant &#945;-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.</a> J. Biol. Chem. 281 (1), 334-340 (2006).</li><li>Kuwahara, T., Koyama, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+reveals+involvement+of+endocytic+pathway+in+neuronal+dysfunction+in+a-synuclein+transgenic.">A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in a-synuclein transgenic.</a> 17 (19), 2997-3009 (2008).</li><li>Su, L. J., Auluck, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Dis. Model Mech. 3 (3-4), 194-208 (2010).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+Caenorhabditis+elegans+in+a+microfluidic+environment.">Electrotaxis of Caenorhabditis elegans in a microfluidic environment.</a> Lab Chip. 10 (2), 220-226 (2010).</li><li>Salam, S., Ansari, A., et al. <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+set+up+to+study+neuronal+degeneration+and+identification+of+neuroprotective+compounds+in+C.+elegans.">A microfluidics set up to study neuronal degeneration and identification of neuroprotective compounds in C. elegans.</a> , (2013).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+sorting+of+Caenorhabditis+elegans.">Electrical sorting of Caenorhabditis elegans.</a> Lab Chip. 12 (10), 1831-1840 (2012).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pulse+direct+current+signals+on+electrotactic+movement+of+nematodes+Caenorhabditis+elegans+and+Caenorhabditis+briggsae.">Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae.</a> Biomicrofluidics. 5 (4), 044116 (2011).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Caenorhabditis+elegans+in+alternating+electric+field+and+its+application+to+their+localization+and+control.">Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control.</a> Appl. Phys. Lett. 96 (15), 153702 (2010).</li><li>van Ham, T. J., Thijssen, K. L., Breitling, R., Hofstra, R. M., Plasterk, R. H., Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+model+identifies+genetic+modifiers+of+alpha-synuclein+inclusion+formation+during+aging.">C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.</a> PLoS Genet. 4, e1000027 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Gabel, C. V., Gabel, H., Pavlichin, D., Kao, A., Clark, D. A., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+mediate+electrosensory+behavior+in+Caenorhabditis+elegans.">Neural circuits mediate electrosensory behavior in Caenorhabditis elegans.</a> J. Neurosci. 27 (28), 7586-7596 (2007).</li><li>Chuang, H. -. S., Raizen, D. M., Lamb, A., Dabbish, N., Bau, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectrophoresis+of+Caenorhabditis+elegans.">Dielectrophoresis of Caenorhabditis elegans.</a> Lab Chip. 11 (4), 599-604 (2011).</li><li>Cronin, C. J., Mendel, J. E., Mukhtar, S., Kim, Y. -. M., Stirbl, R. C., Bruck, J., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+measuring+parameters+of+nematode+sinusoidal+movement.">An automated system for measuring parameters of nematode sinusoidal movement.</a> BMC Genet. 6, 5 (2005).</li><li>Mani&#232;re, X., Lebois, F., Matic, I., Ladoux, B., Meglio, J. -. M. D. i., Hersen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+worms:+C.+elegans+self-sorting+by+electrotaxis.">Running worms: C. elegans self-sorting by electrotaxis.</a> PLoS One. 6 (2), e16637 (2011).</li></ol>

Die mikrofluidischen electrotaxis Assay-Technologie ist zum Patent wurde in den Vereinigten Staaten von Amerika und Kanada eingereicht.

Unter Ausnutzung der Verhaltens-Phänomen erstmals von Gabel und Kollegen und Gebäude auf dem dielektrophoretischen Manipulation Arbeit von Chuang und Kollegen 11,12 beschrieben, stellt unsere Mikrofluidik-basierten electrotaxis Assay eine einfache, robuste und sensitive Methode, um neuronale Aktivität in Worms mit Bewegung als Sonde einen Ausgang. Die Analyse der Parameter der Bewegung ermöglicht quantitative Vergleich zwischen verschiedenen Genotypen. Die Präzision der Fertigung und Mikrokanalstruktur e...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

Der Fadenwurm Caenorhabditis elegans ist ein vielseitiges Modell für biomedizinische Forschung wegen seiner Erhaltung der krankheitsbedingten Gene und Signalwege sowie seiner einfachen Anbau. Mehrere C. elegans Erkrankungsmodellen berichtet worden, wie neurodegenerative Erkrankungen wie Parkinson-Krankheit (PD), welche die Degeneration dopaminerger (DA) Neuronen 1 umfasst. Beide Transgene und neurotoxische Chemikalien verwendet worden, um DA Neurodegeneration und damit verbundene Bewegung Defekte in Würmern zu induzieren, so dass für Untersuchungen der Grundlage der Neurodegeneration und Bildschirme für neuroprotektive Gene und Verbindungen 2,3. Screens in niederen Eukaryoten wie C. elegans stellen ein effizientes und wirtschaftliches Mittel, um Verbindungen und Gene, die neuronale Signalübertragung identifizieren. Herkömmliche Bildschirme sind in der Regel manuell durchgeführt und erzielte durch visuelle Inspektion, folglich sind sie Zeit-consuming und anfällig für menschliche Fehler. Zusätzlich sind die meisten konzentrieren sich auf zellulärer Ebene Analyse unter Ignorierung Fortbewegung, ist das ein besonders wichtiger Parameter für Bewegungsstörungen. Wir haben einen neuen mikrofluidischen Screening-System (Abbildung 1), die quantifiziert und steuert C entwickelt elegans &#39;Fortbewegung mit elektrischen Feldes innerhalb Mikrokanäle Reize. Wir haben gezeigt, dass ein Gleichstrom (DC)-Feld kann robust on-demand Fortbewegung in Richtung der Kathode (&quot;electrotaxis&quot;) 4 induzieren. Umkehrung des Feldes Polarität der Wurm schnell seine Richtung umzukehren als gut. Wir haben auch gezeigt, dass Defekte in dopaminergen und anderen sensorischen Neuronen den Swimming Antwort 5 ändern. Daher kann Anomalien in neuronalen Signalübertragung ermittelt Fortbewegung als read-out werden. Die Bewegung Antwort kann genau quantifiziert werden mit einer Reihe von Parametern wie Geschwindigkeit Schwimmen, Körper Biegeeigenfrequenz und Wendezeit. <p class = &quot;jove_content&quot;&gt; Unsere Arbeit hat gezeigt, dass die Reaktion mit electrotactic Alter variiert. Insbesondere junge Erwachsene zu einem niedrigeren Bereich von elektrischen Feldern zu reagieren und bewegen sich schneller im Vergleich zu 4 Larven. Diese Erkenntnisse führten wir eine neue Mikrofluidikvorrichtung passiv sortieren Würmer nach Alter und Phänotyp 6 entwerfen. Wir haben auch die Reaktion der Würmer gepulsten DC getestet und Wechselstrom (AC) elektrische Felder. Pulsed DC Felder der verschiedenen Arbeitszyklen effektiv erzeugt electrotaxis sowohl in C. elegans und seinen Cousin C. briggsae 7. In einem anderen Experiment immobilisiert symmetrisch Wechselfelder mit einer Frequenz von 1 Hz bis 3 KHz Würmer in den Kanal 8. Umsetzung des elektrischen Feldes in einem mikrofluidischen Umgebung ermöglicht eine schnelle und automatisierte Ausführung des electrotaxis Assay. Dieser Ansatz verspricht hohen Durchsatz genetischen und chemischen Faktoren für Bildschirme erleichternbeeinflussen neuronale Funktion und Lebensfähigkeit.

Eine repräsentative Video von einem Wildtyp-young adult Nematoden electrotaxis und ihre Position und Geschwindigkeit Ausgänge von der Schnecke Tracking-Software sind in Ergänzende Video 1 und Abbildung 3 dargestellt. Die Bewegungsanalyse-Software selbst nicht erkennt die Richtung des Feldes Polarität und die Zeit der Umpolung, sondern müssen diese Informationen von der Quelle Video erhalten werden. Dies könnte mit Hilfe eines Audio-oder visuellen Hinweis in der Video-oder aufzusch...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

Eine halbautomatische mikroelektromechanischen fluidischen Verfahren zur On-Demand-Fortbewegung in induzieren<em&gt; Caenorhabditis elegans</em&gt; Beschrieben. Diese Methode basiert auf dem Phänomen der neurophysiologischen Reaktion auf Würmer milden elektrischen Feldern (&quot;electrotaxis&quot;) in mikrofluidischen Kanälen basiert. Microfluidic electrotaxis dient als schnelle, empfindliche, kostengünstige und skalierbare Technik zum Screening auf Faktoren, die neuronalen Gesundheit.

Mikrofluidik-basierten electrotaxis für On-Demand-Quantitative Analyse von<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis

Research

JoVE Journal

Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

9.7K Aufrufe.  McMaster University. Eine halbautomatische mikroelektromechanischen fluidischen Verfahren zur On-Demand-Fortbewegung in induzieren Caenorhabditis elegans Beschrieben. Diese Methode basiert auf dem Phänomen der neurophysiologischen Reaktion auf Würmer milden elektrischen Feldern ("electrotaxis") in mikrofluidischen Kanälen basiert. Microfluidic electrotaxis dient als schnelle, empfindliche, kostengünstige und skalierbare Technik zum Screening auf Faktoren, die neur...

Serie: Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Verbesserte Visualisierung und quantitative Analyse von Arzneimittelwirkungen mit mikrostrukturierten Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Forschung

Biotechnik

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

Einem mikrofluidischen-basierte Hydrodynamische Trap für einzelne Teilchen

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

Quantitative Locomotion Study of Frei Schwimmen Mikroorganismen mittels Laserbeugung

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Quantitative FRET (Förster Resonance Energy Transfer) Analyse für SENP1 Protease Kinetics Bestimmung

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Drug-induzierte Sensibilisierung der Adenylatzyklase: Assay Straffung und Miniaturisierung für Small Molecule-und siRNA-Screening-Anwendungen

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Einem mikrofluidischen Biochip-basierte Elektrochemische für Markierungsfreie DNA-Hybridisierungsanalyse

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Electro Studium der Lungenkrebszellen unter Verwendung eines Mehrkanal-Zwei elektrischen Feldes Mikrofluidik-Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Mehrfarbenfluoreszenzdetektion für Droplet Mikrofluidik Verwendung von Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

Eine effiziente und reproduzierbare Protokoll für Ablenkung Osteogenesis in einem Rattenmodell führt zu einer funktionalen regenerierten Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Mikrofluidische Plattform für längs-Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...