Forschung wurde durch die NIH Grants EY012031 und EY0182175 und die FM Kirby-Stiftung unterstützt.

Optische Pinzetten zur Manipulation von isolierten Zellen in Kultur Trapping Kräfte der optischen Pinzette aus der Dynamik des Lichts (.; Ashkin et al, 1986 Ashkin, 1991) generiert. Obwohl diese Kräfte leicht Trap Zellen in Suspension, sind sie nicht in der Lage, Zellen, die an einer Oberfläche haften zu bewegen. Daher die Haftung auf der Kulturschale zu dem Punkt, wo Zellen eingeschlossen zu reduzieren, wird das Deckgläschen mit Poly-2-Hydroxyethylmethacrylat (Poly-HEMA) (Sigma Chemical Co., St. Louis, MO), eine ungiftige Verbindung mit Zell-abweisend beschichtet Eigenschaften bisher für die Haftung Studien auf Endothelzellen (Folkman und Moscona, 1978) beschäftigt. Während Poly-HEMA deckt die Hälfte der Kulturschale, die andere Hälfte mit einem Substrat, das Zellwachstum unterstützt beschichtet. In unserem Labor ist Sal-1 als Substrat für die Kultivierung Salamander Netzhautzellen verwendet. Das Verfahren zur Herstellung von Sal-1 und Poly-HEMA beschichtet Gerichte wird nachfolgend beschrieben: Herstellung von Kulturschalen <ol><li> Saubere Deckgläsern (# 1 VWR Scientific Inc., Media, PA) in Säure. Das Deckglas wurde für seine Dicke von nur 0,1 mm, die für die hohe numerische Apertur Ziele für die Fokussierung des Lasers erforderlichen ausgewählt. </li><li> So erstellen Sie eine Zelle repellant Gebiet, lösen 20 mg Poly-HEMA (Sigma Chemical Co., St. Louis, MO) in 1 ml 95% igem Ethanol über Nacht bei Raumtemperatur. Vorzugsweise sollte diese Lösung innerhalb von 1 Monat Zubereitung verwendet werden. </li><li> Prop den Deckgläsern in einer fast senkrechten Position in einer staubfreien Umgebung wie eine Kapuze. </li><li> Legen Sie ein oder zwei Tropfen von Poly-HEMA-Lösung in der Nähe der Spitze des Deckglas aus dem Kunststoff Spitze eines 200 ul Mikropipette. Die Tropfen (ca. 50-100 &amp;mgr; jedem) sollte auf eine Hälfte des Deckglas eingeschränkt werden. Die steilen Hang sorgt die Flüssigkeit auf der Oberfläche des Deckglas auf den Boden, eine dünne gleichmäßige Beschichtung von Poly-HEMA. Lassen Sie die Poly-HEMA beschichtet Deckgläsern in der Motorhaube für 30-60 Minuten bei Raumtemperatur trocknen. </li><li> Markieren Sie die Poly-HEMA Seite der Deckglas mit einem feinen Filzstift an die Kante. </li><li> Drill ein 1 cm großes Loch in den Boden einer 35 mm Kulturschale. </li><li> Kleben Sie die Poly-HEMA beschichtet Deckglas auf den Boden der Kulturschale mit Sylgard 184 (Dow Corning Co., Midland, MI), so dass die Poly-HEMA beschichteten Seite in Richtung der Innenseite der Schale Gesichter. Sicherstellen, dass die Kante des Poly-HEMA-Beschichtung läuft in der Mitte des Loches. Lassen Sie das Geschirr für 1-3 Tage bei Raumtemperatur in einer staubfreien Umgebung trocken. </li><li> Scratch eine Linie auf der Außenseite des Deckglas mit einer Diamant-Spitze Pen nach der Kante des Poly-HEMA-Beschichtung. Dann kratzen zwei sich kreuzenden Linien im rechten Winkel zu etwa 2mm auseinander. Die beiden Schnittpunkte dienen als Referenz-oder Bezugspunkte für die Wiedergabe der gleichen Ausrichtung der Kulturschale auf dem Mikroskoptisch verwendet werden. </li><li> Sterilisieren Sie die Speisen unter UV-Licht über Nacht. </li><li> Um eine Zelle haftenden Hälfte der Kulturschale, Mantel der nicht poly-HEMA Hälfte mit Sal-1, das eine Maus anti-Salamander Antikörper gegen retinale Zellmembranen (freundlicherweise von Dr. Peter MacLeish, Morehouse School of Medicine vorgesehen angehoben enthält erstellen , Atlanta, GA, siehe MacLeish et al (1983)).. Zunächst beschichten Sal-1-Seite mit ca. 100 &amp;mgr; von 0,1 mg / ml sterile Ziege anti-Maus IgG-Antikörper (Boehringer Mannheim Corporation, Indianapolis IN), kümmert sich nicht um die Flüssigkeit über auf das Poly-HEMA Seite zu verschütten. Verlassen Sie für drei Stunden, dann entfernen Sie sie. Waschen Sie die gleiche Fläche mit steriler Ringer-Lösung ein-oder zweimal, dann Platz 100 &amp;mgr; Sal-1 Überstand, kümmert sich nicht um die Flüssigkeit in den Poly-HEMA Seite der Schale zu verschütten. Der erste Antikörper sorgt für Sal-1 bei einer bekannten Konzentration beschichtet ist. </li><li> Inkubieren Sie die Gerichte mit der Sal-1 Nacht. </li><li> Entfernen Sie die Sal-1-Antikörper-Lösung und spülen Sie die Fläche mit sterilen Ringerlösung. Führen Sie 2 ml Medium, um die Schale kurz vor Zell-Beschichtung. </li></ol> Die Zusammensetzung der Amphibien-Ringer-Lösung, Serum-freien Medium Amphibien und Ringer-Lösung für die Enzym-Verdauung sind in MacLeish und Townes-Anderson (1988) und Mandell et al. (1993). Vorbereitung der Zellkulturen Die Zellen in diesem Video verwendet wurden abgeleitet helladaptierte, Erwachsenen-, Wasser-Phase Tiger Salamander (Ambystoma tigrinum), Mess 17-22 cm in der Länge (Charles Sullivan Inc., Nashville, TN), bei 5 ° C gehalten auf einer 12 h: 12 h Hell-Dunkel-Zyklus. Die Protokolle wurden von der Institutional Animal Care genehmigt und Verwenden Committee (IACUC) an der University of Medicine and Dentistry of New Jersey in strikter Übereinstimmung mit den Richtlinien des National Institutes of Health. Das Verfahren zur Vorbereitung der retinalen Zellkulturen werden durch Nachman-Clewner und Townes-Anderson (1996) beschrieben und sind wie folgt: <ol><li> Enthaupten und Mark der Tiere. Entfernen Sie die Augen und sezieren die Hornhaut und Iris. Die Netzhaut ist leicht zu lösen innerhalb der Augenmuschel. Durch die Platzierung einer gebogenen Pinzette unter der Netzhaut, aushöhlen der Netzhaut. Es ist nicht wichtig, wenn die Linse auf die Netzhaut an dieser Stelle angebracht. </li><li> Digest der Netzhaut in 14 U / ml Papain (Worthington, Freehold, NJ) in Salamander Ringer-Lösung für 40 Minuten. </li><li> Vor dem Gebrauch sollte das Papain Ringer-Lösung mit 95% O 2 / 5% CO 2-Gas für 30 Minuten perlen., Dann durch den Durchgang durch einen 0,22-Mikron-Filter (Millipore, Carrigtwohill, Irland) sterilisiert. </li><li> Spülen Sie die Netzhaut mit einer sterilen Salamander Ringer-Lösung dreimal. </li><li> Entfernen Sie die Linsen aus der Ringer-Lösung, dann sanft verreiben der Retina mit einer 3 mm Bohrung Pipette auf eine Zellsuspension zu erhalten. </li></ol> Laser Pinzette Manipulation von isolierten Neuronen <ol><li> Legen Sie die Kulturschale auf dem Mikroskoptisch, Ausrichtung der Schüssel durch Ausrichtung einer Marke auf dem Teller mit einem Bezugspunkt auf der Bühne. Speichern Sie die Koordinaten der Bezugspunkte in den Computer ein. </li><li> Platte Zellen auf beiden Sal-1 und Poly-HEMA Hälften der Schale. </li><li> Lassen Sie die Zellen für ca. 20 Minuten zu regeln, die lang genug ist für die Zellen auf die haftende Seite der Schale an den Antikörper Substrat befestigen. </li><li> Wählen Sie eine Zelle auf der Sal-1 haftende Seite der Schale. Markieren Sie die x-und y-Koordinaten der Bühne einer Position nahe der Zelle. In unserem Fall war der Punkt, etwa 10 um von der primären Dendriten der ausgewählten Zelle. Speichern Koordinaten in den Computer ein. </li><li> Wählen Sie ein zweites Neuron, in unserem Fall einen Photorezeptor, auf der Poly-HEMA Seite der Schale und die Laserpinzette Werkzeug, um es trap. Trapping kann über eine breite Palette von Leistungen erreicht werden. Allerdings setzen wir die Leistung des Lasers bei 10% -20%, die niedrig genug, um Trapping Schutt zusammen mit der Zelle zu vermeiden ist, aber ausreicht, um die Zelle durch das Medium zu transportieren. </li><li> Halten Sie die Zelle, in die Falle, senken Sie die Bühne, so dass die Zelle liegt deutlich über der Oberfläche der Kulturschale und vor allen angeschlossenen Neuronen. Bewegen Sie die Bühne unter Computer-Kontrolle der Zelle, um die zuvor gespeicherten x-und y-Koordinaten zu bringen auf die Sal-1-Seite. Die Bühne Bewegung war in 8-20 um / s eingestellt, aber Höchstgeschwindigkeit sollte empirisch ermittelt werden. Bei der Ankunft am Zielpunkt auf der Sal-1-Seite, heben die Bühne, um die eingeschlossenen Zellen an der Oberfläche der Schale zu bringen. Zell-Platzierung können mit einer Genauigkeit von wenigen Mikrometern hergestellt werden. Lassen Sie die Zelle, die Sal-1 Substrat haften. </li><li> Beziehen digitalisierten Bilder der beiden Zellen in das Paar vor und nach der Paarbildung ist ein nützliches aufzeichnen. </li><li> Pflegen Sie die Zelle paarweise in einem Inkubator. Für Salamander-Zellen, die Schalen in einer feuchten Kammer bei 10 ° C. Die Zellen können per Video Zeitraffer überwacht werden und mit jedem zellbiologischen Technik erforderlich. </li></ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Opt. Lett. 11, 288-290 (1986).</li><li>Clarke, R. J., Hoegnason, K., Brimacombe, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+cells+have+different+target+preferences+in+vitro+as+revealed+by+optical+tweezers.">Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.</a> Mol. Vision. 14, 706-720 (2008).</li><li>Folkman, J., Moscona, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Liu, Y., Cheng, D. K., Soneck, G. J., Berns, M. W., Chapman, C. F., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+localized+cell+heating+induced+by+infrared+optical+tweezers.">Evidence of localized cell heating induced by infrared optical tweezers.</a> Biophysical Journal. 68, 2137-2144 (1995).</li><li>MacLeish, P. R., Barnstable, C. J., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Proc. Natl. Acad. Sci. USA. 80, 7014-7018 (1983).</li><li>MacLeish, P. R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synapse+formation+among+major+classes+of+adult+salamander+retinal+neurons+in+vitro.">Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.</a> Neuron. 1, 751-760 (1988).</li><li>Mandell, J. W., MacLeish, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Townes-Anderson+E.+Process+outgrowth+and+synaptic+varicosity+formation+by+adult+photoreceptors+in+vitro.">Townes-Anderson E. Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.</a> J. Neurosci. 13, 3533-3548 (1993).</li><li>Nachman-Clewner, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-induced+remodelling+and+regeneration+of+the+ribbon+presynaptic+terminal+in+vitro.">Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.</a> J. Neurocytol. 25, 597-613 (1996).</li><li>Townes-Anderson, E., St Jules, R. S., Sherry, D. M., Lichtenberger, J., Hassanain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+retinal+neurons+by+optical+tweezers.">Micromanipulation of retinal neurons by optical tweezers.</a> Mol. Vis.. 4, (1998).</li></ol>

Licht hat an Dynamik, und wenn ein Lichtstrahl gebrochen wird, wie es durch eine Zelle durchläuft, eine Kraft ist erforderlich, um die Richtung des Impulses zu ändern. Wegen des Gesetzes von der Erhaltung des Impulses, muss eine Kraft in die entgegengesetzte Richtung, die ihrerseits wieder reagieren auf eine Zelle. Ashkin (1991) zeigten, dass die Kraft, die durch einen Laserstrahl durch ein Mikroskopobjektiv fokussiert generiert wird eine Zelle zur Mitte des Fokus zu verschieben. Auch wenn ein Laserstrahl erzeugt nur ein paar Pikonewton v...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>25mm circle No.1 coverglass</td>
<td>&nbsp;</td>
<td>VWR Scientific Inc., Westchester, PA</td>
<td>48380 080</td>
<td>&nbsp;</td>
<tr>
<td>poly-2-hydroxyethylmethacrylate (poly-HEMA)</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co., St Louis, MO</td>
<td>P-3932</td>
<td>Dissolve in 95% ethanol</td>
</tr>
<tr>
<td>Goat anti-mouse IgG antibody</td>
<td>&nbsp;</td>
<td>Chemicon International, Temecula CA</td>
<td>AP181</td>
<td> 1mg in 1ml, dilute 10x for use</td>
</tr>
<tr>
<td>Sal-1 supernatant containing mouse anti-salamander antibody</td>
<td>&nbsp;</td>
<td>generously provided by Dr. Peter MacLeish</td>
<td>&nbsp;</td>
<td>Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA</td>
</tr>
<tr>
<td>3 mm bore 5ml pyrex disposable pipets</td>
<td>&nbsp;</td>
<td>Corning Inc., Corning NY</td>
<td>7078A-5</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes 35mm x 10mm</td>
<td>&nbsp;</td>
<td>Corning inc., Corning NY</td>
<td>430165</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 silicone elastomer kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Optical tweezers-microtool or laser tweezers</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 W continuous wave diode laser of 980nm wavelength</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Axiovert 100 inverted light microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40x oil immersion plan neofluor objective lens</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>Numerical aperture (N.A. 1.3)</td>
</tr>
<tr>
<td>Black and white CCD camera</td>
<td>&nbsp;</td>
<td>Sony Corporation, Tokyo, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer and joystick with software</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc.</td>
<td>&nbsp;</td>
<td>for controlling a motorized stage</td>
</tr>		</tbody>
		</table>
		</div>

using laser tweezers for manipulating isolated neurons in vitro

In diesem Papier und Video beschreiben wir die Protokolle in unserem Labor verwendet werden, um die Ausrichtung Präferenzen der regenerierenden Zellen Prozesse der erwachsenen Netzhautneuronen in-vitro-Studie. Verfahren zur Vorbereitung der retinalen Zellkulturen beginnen mit der Präparation, die Verdauung und Zerreiben der Netzhaut, und enden mit der Beschichtung von isolierten Zellen der Netzhaut auf Gerichte speziell für den Einsatz mit Laser-Pinzette gemacht. Diese Gerichte sind in eine Zelle Klebstoff Hälfte und eine Zelle repellant zwei Hälften geteilt. Die Zelle Klebeseite ist mit einer Schicht von Sal-1-Antikörper, die ein Substrat, auf denen unsere Zellen wachsen bieten beschichtet. Andere Klebstoff Substrate könnte auch für andere Zelltypen verwendet werden. Die Zelle repellant Seite ist mit einer dünnen Schicht aus Poly-HEMA beschichtet. Die Zellen auf der Poly-HEMA Seite der Schale überzogen sind mit dem Laser Pinzette gefangen, transportiert und dann neben der Sal-1-Seite in eine Zelle gelegt, um ein Paar zu schaffen. Formation von Zell-Gruppen jeder Größe sollte mit dieser Technik möglich. &quot;Laser-Pinzette-gesteuerte Mikromanipulation&quot; bedeutet, dass der Prüfer können wählen, welche Zellen sich zu bewegen, und den gewünschten Abstand zwischen den Zellen können standardisiert werden. Da der Laserstrahl geht durch transparente Oberflächen der Kulturschale, sind Zelle Auswahl und Platzierung in einem geschlossenen, sterilen Umgebung gemacht. Die Zellen können per Video im Zeitraffer überwacht werden und mit jedem zellbiologischen Technik erforderlich. Diese Technik kann helfen, Untersuchungen von Zell-Zell-Interaktionen.

Watch this Scientific Journal Video about Using Laser Tweezers For Manipulating Isolated Neurons In Vitro at JoVE.com

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Dieses Video beschreibt die Manipulation von kultivierten Neuronen mittels Laser-Pinzette in vitro.

Mit Laser-Pinzette zur Manipulation isolierter Neuronen In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

using-laser-tweezers-for-manipulating-isolated-neurons-in-vitro

Research

JoVE Journal

Biology

10.3K Aufrufe.  University of Medicine and Dentistry of New Jersey - UMDNJ. Dieses Video beschreibt die Manipulation von kultivierten Neuronen mittels Laser-Pinzette in vitro.

Serie: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Mess-Exocytose in Neuronen mit FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Forschung

Biologie

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

Silizium-Mikrochips zur Manipulation Zell-Zell-Interaktion

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Mit Micro-Electro-Mechanical Systems (MEMS) zu Diagnose-Tools entwickeln

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

In-vitro-Assays mit Aggregation hyperphosphoryliertem Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

Manipulation der Murine Lakrimal

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Die Maus isoliert perfundierten Nieren-Technik

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analyse von Zellsuspensionen isoliert von festen Geweben von Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.