Funding was provided by the Joint Science and Technology Office &#8211; Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).

Platten 20.000 differenziert Maus-ES-Zellen (MES) / Well in einer 96-Well Poly-D Lysin beschichteten Platten und aufrechtzuerhalten in Motoneuronen-terminalen Differenzierung Medien für 5-7 Tage. 1. Verbindung Verwaltung und Intoxikation mit BoNT / A Führen Sie alle der folgenden Arbeit in einem BSL2 Gehäuse, um die Einhaltung CDC / NIH-Richtlinien zu halten. <ol><li> Herstellung von 10 mM Stammlösungen der einzelnen Bibliotheksverbindung in 100% DMSO in 96-Well-Polypropylenplatten. Nutzen Sie die 10 mM Stamm um eine Zwischenverdünnungsplatte, die 10-fach größer als die gewünschte Screening Konzentration vorzubereiten. Bereiten 100 uM Zwischenplatte durch den Verzicht 10 mM Stamm in 96-Well-Platte und verdünnen Sie es mit Kulturmedien bis 100 &amp; mgr; M. Verzichtet werden 10 &amp; mgr; l der Verbindungen auf 90 ul Medium auf den Zellen 11. Testen Sie die Verbindungen bei 10 uM Endkonzentration und sorgen für die endgültige Prozentsatz der DMSO 0,5% nicht überschreiten. </li><li> Führen Sie alle Bolzenies, die lebenden Zellen und aktives Toxin unter Biosicherheitsstufe 2 Bedingungen zu nutzen. Ersetzen Sie die alten Medien in den 96-Well-Platten mit 80 ml frisches terminalen Differenzierung Medien mit einem 12-Kanal-Handpipette. Führen Sie Aspiration und verzichten Schritte durch Reihen, um die Exposition der Zellen gegenüber der Umgebungsluft zu vermeiden, ohne Medien. </li><li> Vorzubehandeln Zellen mit Verbindungen vor der Applikation von Toxin durch Zugabe von 10 ul 10x-Verbindungen von der Quelle-Platte unter Verwendung einer 12-Kanal Pipette, gefolgt von Mischen durch Absaugen (zweimal). Für jede 96-Well-Platte, behandeln zwei Spalten mit 6 Vertiefungen mit 10x DMSO in den Medien verdünnt, um als High-Signal und niedrige Signalsteuerungen dienen. Die Säule ohne BoNT-Behandlung weist das höchste Niveau der vollen Länge SNAP-25 (High-Signal-Steuerung). Behandeln Sie die anderen Säule mit BoNT allein (ohne Verbindungen) als Low-Signal Kontrolle. Verwenden Sie niedrige Steuer, um die Effizienz von BoNT Spaltung SNAP-25 und dessen Nachweis mit der BACS-Antikörper zu beobachten (Abbildung 2). </li><li> Inkubieren Zellen mit Verbindungen für 30 min bei 37 ° C und 6% CO 2 in den Zellkulturbrutschrank. </li><li> Vorbereitung Botulinum Neurotoxin A aus einer 1 mg / ml kommerziellen Quelle in Phosphat-gepufferter Salzlösung (PBS) auf eine Arbeitsstamm 10x durch Verdünnen mit terminalen Differenzierung Medien. </li><li> Initiate Intoxikation durch Zugabe von 10 ul 10x BoNT / A Lager in für die Behandlung und Low-Signal steuert mit einer Mehrkanalpipette (Abbildung 2) Kavitäten pipettieren. Behandeln Sie die so hoch Steuerung mit Medien allein Kavitäten pipettieren. </li><li> Dekontaminieren alle Kunststoffwaren sowie anderen Einmal die in Kontakt mit BoNT / A während der Studie durch Eintauchen in 0,825% Hypochlorit-Lösung in Wasser für mindestens 20 Minuten kommt. Führen Sie alle in der biologischen Sicherheit Schränke und dekontaminieren Arbeitsbereiche vor und nach Experimenten mit 5% Detergens Desinfektionsreiniger wie MicroChem Lösung. </li><li> Beschriften Sie die Platten eindeutig Toxin Gebrauch und Incub bezeichnenaß Platten mit 1 nM / L BoNT / A für 4 h bei 37 ° C und 6% CO 2 in einem Zellkulturbrutschrank behandelt. Anzeige entsprechende Beschilderung an Kollegen, dass Toxin in den Einsatz im Labor zu informieren. </li><li> Stopp BoNT / A proteolytischen Spaltung durch Methanol-Fixierung. Entfernen Sie das Medium Toxin und Verbindungen, die aus jeder Vertiefung mit einer Mehrkanalpipette und entsorgen in 10% Bleichlösung. Hinzufügen eiskaltem Methanol (100 ul), die direkt in jede Vertiefung. </li><li> Platten für 15 min bei Raumtemperatur (RT) inkubieren, damit Fixierung auftritt. </li><li> Nach der Fixierung sicher zu handhaben, die verworfen Reagenzien (legte neutralisiert) mit Sicherheitsstufe 1 Protokolle und entsorgen entsprechend. </li><li> Entsorgen Methanol und verwenden 100 ul PBS, um die Zellen zu waschen und vor deinen Durst sie Immunfärbung. Führen Sie zwei zusätzliche PBS Wäschen. </li><li> Nach dem letzten Waschen, lassen PBS in den Vertiefungen, Dichtungsplatten mit Mikroklebefolie und lagern bei 4 ° C bis zur Analyse. Store-Platten bei 4 ° C for mehrere Tage. </li></ol> 2. Immunfärbung Die Immunfärbung Prozedur ist eine arbeitsintensive, mehrstufigen Vorgang, der sich wiederholenden Reagenz Abgabe / absaugen Zyklen und umfangreiche Plattenwäsche, die der mögliche Einführung von signifikanten Intra und Interplate Variabilität führen kann, schließt. Eine halbautomatische Ansatz angewendet, um die Zeit des Laborpersonals speichern Assay Durchsatz zu erhöhen und die Immunfärbung Variabilität zu minimieren. <ol><li> Verwenden Sie eine automatisierte Arbeitsstation mit einem 96-Loch-Kopf ermöglicht das Übermengen von bis zu 250 &amp; mgr; l und integriert mit einem Roboter-Greifarm, um Laborgeräte in verschiedenen Orten auf dem modularen Deck (siehe Material und Ausrüstung) für dieses Protokoll zu bewegen ausgestattet. <ol><li> Die modulare Plattform wird entworfen, um verschiedene Arten von Laborgeräte hosten und kann bis zu 11 Artikel zu einem beliebigen Zeitpunkt zu unterstützen. Das automatisierte Mikro Handler ist mit einem Dual-Magazin Mikroplatten-Stacker, um zu halten ausgestattet undmanipulieren bis zu 50 Platten pro Zyklus. </li><li> Die automatisierte Arbeitsstation innerhalb einer Klasse II Biosicherheitswerkbank für die Laborautomatisierung Ausrüstung, die der Sterilisation und Sicherheit bieten entfernt. Für Immunfärbeautomaten wird das Deck angeordnet ist, wie in 3 gezeigt, um den Zugang Reagenz nach Bedarf und ohne menschlichen Eingriff zu ermöglichen. </li></ol></li><li> Pre-load-Platten, die fixierten Zellen in das Plattenmagazin und Transfer zum Deck wie für Liquid Handling benötigt. Nutzen Sie die 96 Pipettenkopf mit 250 ul Tipps versehen, um PBS aus den Vertiefungen auf 10 ul absaugen. Durchdringbar Zellmembranen zu Antikörper-Bindung an intrazelluläre Ziele mit Permeabilisierung Puffer (0,1% Triton-X 100) zu erleichtern. Verzichtet Permeabilisierung Puffer (100 ul) in jede Vertiefung der Platten vor der Rückkehr zu dem zweiten Speichermagazin der Mikrostapel für eine 15 min Inkubation bei Raumtemperatur (RT). </li><li> Entfernen Permeabilisierung Puffer und perform Waschen der Platte mit PBS (zweimal), wie zuvor in Schritt 1.13. </li><li> Nach dem letzten Waschschritt entfernt das PBS-Puffer zubereitet, und 100 &amp; mgr; l Blockierungspuffer, enthaltend 1% Pferdeserum und 0,1% Tween 20 in PBS in alle Mikrotiterplatten, bevor er sie an den Plattenmagazin 1 h Inkubation bei RT. </li><li> Verwenden Sie zwei primäre Antikörper für Immunfärbung: einen polyklonalen Kaninchen-Anti-Maus-Antikörper BACS, für den Nachweis von Volllängen-SNAP-25 und einem monoklonalen Maus-Anti-III Tubulin-Antikörper zum Nachweis von Neuronen (siehe Material und Ausrüstung). Nach 60 min Inkubation entfernen Blocking-Puffer und 50 ul verzichtet 4UG / ml des Antikörpers GA und 0,5 ug / ml III-Tubulin in Blockierungspuffer in alle Platten. </li><li> Inkubieren für 1 h bei RT Entfernen der primären Antikörper und wasche 3mal mit 100 ul PBS, enthaltend 0,05% Tween (PBST). </li><li> Verwenden eines Anti-Maus-IgG mit Alexa Fluor 647 markiert die -III-Tubulin und Anti-Kaninchen-IgG mit Alexa F markiert visualisierenluor 568 die BACS-Antikörper sichtbar zu machen. Vorbereitung sowohl Antikörper als 2 &amp; mgr; g / ml-Verdünnung in Blockierungspuffer und abzug 50 ul der Mischung in jeder Vertiefung. HINWEIS: Die sekundären Antikörper für die Immunfärbung ausgewählt werden mit verschiedenen Fluorophoren markiert, um den Nachweis der emittierten Fluoreszenzlicht bei unterschiedlichen Wellenlängen mit unterschiedlichen Kanälen innerhalb des Detektors der hohen Gehalt Imager ermöglichen. </li><li> Inkubation für 1 h bei RT. Saugen Sie die Sekundärantikörper und 3x waschen mit 100 ul PBST. </li><li> Nach dem letzten Waschen werden 100 &amp; mgr; l PBS, das Hoechst-Farbstoff (32 uM), um die DNA für die Kerne Erfassungs färben. </li><li> Dichten Sie die Platten mit einer lichtundurchlässigen Folie, um die Fluorophore von Photobleaching zu schützen. Platten können bei 4 gelagert werden &#160; ° C wochenlang ohne signifikanten Verlust des Signals. </li></ol> 3. Imaging HINWEIS: Führen Sie die Bildaufnahme mit High Content Imaging System (siehe Materialien und Ausrüstung). <ol><li> Spezifikation der High Content Imager Definitionen: <ol><li> Wählen Plattentyp, Layout, Anzahl der Felder, Ziel: Greiner u klar, 96-Well-Format, 16, 20X Wasser auf. </li><li> Kanäle auswählen: Kern Anregungs EX: 405 nm als Kanal 4; Zytoplasma EX: 644 nm als Kanal 2; Antigen-spezifische Antikörper Kanal EX: 561 nm, wie Kanal 3 und Set up Erregerleistung jedes Lasers, Setup-Experiment als 2 Expositionen, Belichtung 1 mit einer Anregung bei 644 nm, Belichtung 2 mit zwei Anregungen bei 405 nm und 561 nm. Wählen Binning als BIN2. </li><li> Verwenden Sie qualitativ Kontrollvertiefungen für Belichtungsoptimierung mit maximaler Intensität wie SNAP-25-Kanal und geringe Kontrollvertiefungen für minimale Intensität. </li><li> Einstellung der Belichtung, so daß die Intensität von jedem Kanal beträgt etwa die Hälfte der Sättigungspegel (2000-2500 relativen Einheiten). </li><li> Identifizieren Sie die optimale Z-Ebene auf die Zellmaskenkanal mittels Delta-Korrektur-Skript. Siehe FiAbbildung 5 für Ergebnisse nach Immunfärbung und Imaging. Exportieren von Daten an den Server, wo die Bildanalyse-Software ist wohnhaft. </li></ol></li></ol> 4. Bildanalyse (Abbildung 6) HINWEIS: Die folgenden Schritte beschreiben die Anwendung der Columbus Software-Algorithmen. <ol><li> Wählen Sie einen geeigneten Algorithmus zur Segmentierung des Primärobjekte (Kerne). Falls erforderlich, passen Sie Hintergrundschwelle und Kontrast-Parameter. Das Columbus-Software bietet 4 Kerne Nachweismethoden. Wählen Sie die Methode, dass die Segmentkerne genau durch Sichtprüfung segmentiert Kerne (in B ild 6a Verfahren M ausgewählt ist). </li><li> Wählen Sie das Neuriten-Algorithmus (CSIRO Neuriten Analyse 2) zum Segment der Neuriten Falls erforderlich, passen Sie Hintergrundschwelle und Kontrast-Parameter in den Hintergrund zu entfernen. Siehe Abbildung 6b für Parameter verwendet, um Neuriten erkennen. </li><li> Identifizieren Sie die Neuriten Regionen (Neuriten Segmente) based auf Sekundärobjekte (Neuriten) unter Verwendung eines Pixel -3 Expansion des β-III-Tubulin-Kanal (Alexa Flour 640) als Maske (6c). </li><li> Berechnen Intensität der Signalkanäle (SNAP-25 (Alexa Flour 568) für das Neuriten-Region und β-III-Tubulin-Kanal (6d &amp; 6e). </li><li> Wählen Sie die gewünschten Parameter für den Export, wie Neuritenlänge, mittleren Intensitäten des SNAP-25, β-III-Tubulin, Kernzahl, Neuriten Segmentnummer usw. (6f). </li><li> Übertragungsplatten aus Plattenstapler mit dem Roboter und Last in die High Content Imager für die Bilderfassung. </li></ol> 5. Datenanalyse: Um die Robustheit des Assays entwickelt beurteilen, Berechnen der folgenden Parameter von der Plattenbasis Experiments. <ol><li> Signal (S) in den Hintergrund (B): S / B = mittlere Intensität hohe Signalsteuerung) / mittlere Intensität niedrige Signalsteuerung </li> &lt;li&gt; Variationskoeffizient (CV) von Signal und Hintergrund (um die Einheitlichkeit des spezifischen Signals messen): CV = (Standardabweichung (SD) / Mittelwert der Stichprobe) * 100 (%), um die Variabilität in Liquid Handling bestimmen, Reagenzien usw. </li><li> Signal to Noise: S = (mittlere Intensität des Signals - mittlere Intensität niedrige Signalsteuerung) / SD niedriger Steuer <ol><li> Der Z-Faktor &quot;Berechnen mit Intoxikation von einer Hälfte einer 96-Well-Platte und einem Mock Rausch der anderen Hälfte. Z &#39;= 1-3 * (SD hoher Signalsteuerung + SD der niedrigen Signalsteuerung) / (Mittelwert der hohen Signalkontrolle - Mittel der niedrigen Signalsteuerung). Für die weitere Analyse der Screening-Daten, zu normalisieren einige der Primärrohstoffe Parameter auf einem Teller Grundlage zum Vergleich zwischen den Platten und zwischen den verschiedenen Tagen der Experimente zu ermöglichen. </li></ol></li><li> Prozent der Spaltung, was die Reduzierung des Signals mit voller Länge SNAP-25 durch spezifische Antikörper nachgewiesen (BACS) zugeordnet:% Dekolleté = 100% * (mean Intensität von Hochsignalsteuerung - Intensität der Probe) / mittlere Intensität hohe Signalsteuerung. </li><li> Prozent der Inhibition der Spaltung:% Hemmung = 100% * (Intensität der Probe - mittlere Intensität niedrige Signalsteuerung) / (mittlere Intensität hohe Signalsteuerung - mittlere Intensität niedrige Signalsteuerung). </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescent+sensors+to+detect+botulinum+neurotoxin+activity+in+vitro+and+in+living+cells.">Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 14701-14706 (2004).</li><li>Stahl, A. M., Adler, M., and, C. B. M., Gilfillan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerating+botulism+therapeutic+product+development+in+the+Department+of+Defense.">Accelerating botulism therapeutic product development in the Department of Defense.</a> Drug Development Research. 70, 303-326 (2009).</li><li>Nuss, J. E., 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=Development+of+cell-based+assays+to+measure+botulinum+neurotoxin+serotype+A+activity+using+cleavage-sensitive+antibodies.">Development of cell-based assays to measure botulinum neurotoxin serotype A activity using cleavage-sensitive antibodies.</a> Journal of biomolecular screening. 15, 42-51 (2010).</li><li>Opsenica, I., 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+chemotype+that+inhibits+three+unrelated+pathogenic+targets:+the+botulinum+neurotoxin+serotype+A+light+chain,+P.+falciparum+malaria,+and+the+Ebola+filovirus.">A chemotype that inhibits three unrelated pathogenic targets: the botulinum neurotoxin serotype A light chain, P. falciparum malaria, and the Ebola filovirus.</a> Journal of medicinal chemistry. 54, 1157-1169 (2011).</li><li>Opsenica, I., 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=The+synthesis+of+2,5-bis(4-amidinophenyl)thiophene+derivatives+providing+submicromolar-range+inhibition+of+the+botulinum+neurotoxin+serotype+A+metalloprotease.">The synthesis of 2,5-bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease.</a> European journal of medicinal chemistry. 53, 374-379 (2012).</li><li>Nuss, J. 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Krishna P Kota is an employee of Perkin Elmer Inc. Waltham, MA, that produces instruments and software used in this manuscript. The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services, the U.S. Department of Defense, the U.S. Department of the Army, or the institutions and companies affiliated with the authors.

Die hohe Wirksamkeit von Botulinum Neurotoxine und der relativen Leichtigkeit ihrer Bewaffnung hat in ihrer Einstufung als Kategorie A (höchste Priorität) biothreat Agenten von den US Centers for Disease Control and Prevention geführt. Leider gibt es keine von der FDA zugelassenen Therapeutika zur BoNT-Intoxikation zu begegnen, nachdem das Toxin von den Motoneuronen verinnerlicht worden. Alle druggable Mechanismus, der neuronalen Wiederaufnahme von BoNT-Intoxikationen fördert könnte in Richtung der Entwicklung eine...

Das Bakterium Clostridium botulinum produziert Botulinum Neurotoxin, einem der potentesten biologischen Giftstoffen auf den Menschen bekannt 1. Es gibt 7 verschiedene BoNT-Serotypen (BoNT / AG). BoNT / A-Gall induzieren die Lähmung an der neuromuskulären Synapse durch SNARE-Komplex Proteolyse 2,3. SNARE Proteolyse verhindert Neurotransmitter-Vesikel-Membranfusion und damit Blöcke Exozytose von Neurotransmittern 4. Die spezifische SNARE Ziel ist abhängig von der jeweiligen BoNT Serotyp im Rausch Prozess beteiligt. BoNT / A und BoNT / E spalten SNAP-25 in der Erwägung, BoNT / C spaltet sowohl SNAP-25 und Syntaxin 5. Die verbleibenden Serotypen spalten Synaptobrevin (auch Vesikel-assoziiertes Membranprotein (VAMP) genannt. BoNT / A wurde für die Assay-Entwicklung gewählt, da es einen hohen Anteil von natürlich vorkommenden Botulismus verantwortlich und hat die längste Wirkdauer. 6 die Entwicklung des kleinen Moleküls Therapeutika gegen BoNT / A ist ein wichtiges Ziel fürunsere Wirkstoffforschungsprogramm und hat die traditionelle Zielbasierte Methoden genutzt, um aktive Zentrum proteolytischen Inhibitoren 7 identifizieren, 8-10. Jedoch wird die Schaffung von aktiven Zentrum Inhibitoren mit breiten Wirkungsspektrum gegen mehrere Serotypen und Postexpositions Wirksamkeit wahrscheinlich schwierig sein. Daher haben wir eine innovative, phänotypische Wirkstoffforschungsansatz, BoNT SNAP-25-Spaltung als funktionelle Endpunkt an kleine Moleküle, die BoNT-vermittelten motorischen Neurons Intoxikation blockieren identifizieren können verwendet implementiert. SNAP-25 ist für die Freisetzung von Neurotransmittern erforderlich, wie Abbau von SNAP-25 ist prädiktiv für Lähmung und Letalität in vivo. Zum Beispiel könnten zellbasierte Screening zur Entdeckung neuer Modulatoren zellulärer Faktoren für Toxin Inaktivierung oder Hemmung der Toxin-Bahn-Innere Zielzellen verantwortlich führen. Ein wichtiger Faktor bei der phänotypischen Assay-Entwicklung ist die Auswahl der physiologisch relevanten biologischen Modellen. We und andere haben embryonalen Stammzellen (ES) Zellen abgeleiteten Motorneuronen, welche die immunologischen Charakter des primären Motoneuronen zu rekapitulieren, einschließlich der Expression von SNAP-25 11- 13 beschrieben. Wichtig ist, dass diese zellulären Systeme sind sehr empfindlich auf BoNT / A-Intoxikation und zeigen Dosis-abhängige Spaltung von SNAP-25 in Reaktion auf ansteigende Konzentrationen des Toxins 11,12. Das differenzierte Motorneuronen sind ebenfalls in Mengen, die ausreichend für die Hochdurchsatzanalyse basiert Platte sind, und erlaubt die Konstruktion einer Reihe von zellulären Assays hergestellt. Die phänotypische Assay ist ein Immunfluoreszenzverfahren, das zwei verschiedene Antikörper zur Spaltung der endogen exprimierten voller Länge SNAP-25 während der BoNT / A Rausch der Maus Motor Neuron Kultur quantifizieren. Ein carboxylterminalen BoNT / A-Spaltung empfindlichen (BACS) Antikörper, der nur in voller Länge SNAP-25 erkennt erlaubt die Beurteilung von BoNT / A vermittelten Proteolyse von SNAP-25Expression in der Maus Motoneuronen 10. Ein schematisches Diagramm des HCl-Assay ist in 1 dargestellt.

<table border="0" cellpadding="0" cellspacing="0" width="834">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Name of Reagent/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Botulinum neurotoxin A&#160;</td>
			<td style="width:180px;">Metabiologics</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;">No catalog number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microtitre plates</td>
			<td style="width:180px;">Greiner&#160;</td>
			<td style="width:119px;">655946</td>
			<td style="width:280px;">Poly-D-Lysine 96-well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BACs antibody</td>
			<td style="width:180px;">Lampire Biological</td>
			<td style="width:119px;">NA</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microchem</td>
			<td style="width:180px;">National&#160;</td>
			<td style="width:119px;">0255</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Methanol</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">A412-20</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Formaldehyde</td>
			<td style="width:180px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Horse serum</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">16050</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">PE JANUS MDT Mini Automated Workstation</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">AJMDT01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Opera</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Triton X 100</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">9002-93-1</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BIII tubulin antibody</td>
			<td style="width:180px;">R&#38;D Systems</td>
			<td style="width:119px;">BAM1195</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween 20</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:119px;">P1379-1L</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342dye&#160;</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">3570</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antimouse IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A21236</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Anti rabbit IgG</td>
			<td style="width:180px;">Invitrogen</td>
			<td style="width:119px;">A10042</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Columbus Image analysis software</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 2.4</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Spotfire</td>
			<td style="width:180px;">Perkin Elmer</td>
			<td style="width:119px;">Ver 5.5</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Clorox bleach</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:119px;">18-861-284</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PlateStack</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td style="width:280px;"></td>
		</tr>
	</tbody>
</table>

a high content imaging assay for identification of botulinum neurotoxin inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Daten aus hohen und niedrigen Kontrollen erstellt zwei verschiedene Populationen mit der Differenz zweier Mittelwerte von mehr als 3 Standardabweichungen (7A). Das Ziel des Screenings ist, um die Verbindungen in der Probenpopulation mit Werten näher positive Kontrollpopulation zu finden, unter der Annahme einer Normalverteilung innerhalb der Probenpopulation (7B, (i)). Datenpunkte, die drei Standardabweichungen über dem Mittelwert liegen werden als statistisch von dem Lärm und als ak...

Watch this Scientific Journal Video about A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors at JoVE.com

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Ein hoher Gehalt Imaging Assay zur Identifizierung von Botulinum Neurotoxin-Inhibitoren

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

a-high-content-imaging-assay-for-identification-botulinum-neurotoxin

Research

JoVE Journal

Neuroscience

8.5K Aufrufe.  Perkin Elmer Inc.. Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Serie: A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Ein organotypischen Assay für High-Resolution Time-Lapse Imaging neuronaler Migration in der postnatalen Gehirn

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Forschung

Neurowissenschaften

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Hochauflösende Functional Magnetic Resonance Imaging Methods for Human Mittelhirn

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Funktionelle Bildgebung Auditory Cortex bei erwachsenen Katzen mit Hochfeld-fMRI

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Isolierung und Quantifizierung von Botulinum Neurotoxin aus komplexen Matrices Mit den Botest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Hohe Auflösung<em&gt; In-vivo-</em&gt; Manuelle Segmentierung Protokoll für Menschen Hippocampus Teilfelder Verwenden von 3T Kernspintomographie

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

Nicht-invasive Parenchymale, Gefäß- und Stoffwechselhochfrequenz-Ultraschall und Photoakustische Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Hochauflösende Strukturkernspintomographie des menschlichen Subcortex<I&gt; In-vivo-</I&gt; Und Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Hochdurchsatz-Analyse des Bewegungsapparates Verhaltens in der Drosophila -Insel-Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Assay-Entwicklung für hohe Content Quantifizierung der Sod1 mutierten Proteins Aggregatbildung in lebenden Zellen

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.