Wir möchten Philippa Warren für ihre freundliche Hilfe danken.

1. Boundary-Test: <ol><li> Grundlegende Zubereitung: Chamber Slides sind mit Poly-D-Lysin über Nacht und sterile Objektträger beschichtet sind, bevor das Experiment vorbereitet. Medium wird hergestellt unter Verwendung Dulbecco modifiziertem Eagle-Medium (DMEM) mit 10% fötalem Kälberserum (FCS) und 1% Penicillin-Streptomycin-Fungizon (PSF). Auch eine Flasche Calcium / Magnesium freie Hanks balanced salt solution (HBSS) hergestellt. </li><li> Primäre Ratten-Schwann-Zellen in Kolben kultiviert werden für 3 Minuten mit 0,1% Trypsin Trypsin behandelt. Primäre Ratten-Astrozyten Kultur ist für 5 Minuten mit 0,1% Trypsin Trypsin behandelt. Trypsin wird durch Zugabe von DMEM, ergänzt mit 10% FCS und 1% PSF inactived. </li><li> Trypsiniert Schwann-Zellen und Astrozyten übertragen werden zu 15 mL Falcon-Röhrchen zu trennen und zentrifugiert bei 300G für 5 Minuten. </li><li> Schwann-Zellen und Astrozyten sind an der Dichte von 2 x 10 6 resuspendiert (siehe Abschnitt repräsentative Ergebnisse für die Unterschiede in der Zelldichte). </li><li> 50 uL Tropfen Schwann Zellsuspension wird an einem Ende der PDL beschichtete Kammer schieben Vertiefungen gegeben. Ein Glas Streifen wurde verwendet, um Abstrich der Tropfen in Richtung der Mitte der Kammer auf eine gerade Kante zu erzeugen. (Mit einem Glasschneider, sind ein rechteckiges Deckgläser längs so, dass es die Breite der Kammer-Objektträgern für Boundary-Assays verwendet fit cut) </li><li> Ein zweiter Tropfen 50 ul Astrozyten Suspension wurde am gegenüberliegenden Ende des gleichen gut aufgestellt und verschmiert in Richtung Zentrum, um eine gerade Kante parallel zur ersten Drop-Abstrich mit einem kleinen 0,2 mm Abstand zwischen ihnen zu schaffen. </li><li> Die Kammer Folien mit den Tropfen in den Inkubator bei 37 º C für 2 Stunden. </li><li> Nach 2 Stunden wurden die Kulturen mit DMEM gewaschen, um nicht angebracht Zelltrümmer und frisches Medium mit DMEM / 10% FCS / 1% PSF aufgenommen zu entfernen. Hinweis: DMEM / 10% FCS / 1% PSF mit Forskolin (2 um) und Bovine Hypophysenextrakt (BPE) (10 ug / mL) stimuliert Schwann-Zellen ergänzt werden kann. BPE liefert die Wachstumsfaktoren notwendig, um langfristig Schwann Zellkultur ermöglichen und Forskolin erhöht die zyklische Adenosinmonophosphat Ebenen innerhalb der Schwann-Zellen zur Proliferation induzieren </li><li> Die Zellen werden für ca. 8-10 Tage bei 37 ° C, 7% CO 2-Inkubator. Das Medium benötigt, um alle 3 Tage gewechselt werden. Die beiden Zellen Fronten stößt irgendwann einander und eine scharfe Grenze zwischen den beiden Zelltypen zu bilden. Zu diesem Zeitpunkt Experimente durchgeführt werden können analysiert Faktoren in Grenzen Bildung beteiligt. </li><li> Co-Kultur kann fest mit 4% Formaldehyd und Schwann-Zellen mit anti-P75-Antikörper und Astrozyten mit anti-GFAP Antikörper gegen die Zellen in Co-Kultur identifizieren immungefärbt werden. P75 Antikörper erkennt eine geringe Affinität NGF-Rezeptor auf Schwann-Zellen, die Astrozyten fehlt. Anti-GFAP Antikörper erkennt Gliazellen firbrillary sauren Protein, das an Astrozyten Zytoplasma lokalisiert. (Siehe Abbildung 1A für repräsentative Grenze) </li></ol> 2. Migration Assay: <ol><li> 4 gut Platten (15mm Bohrlöcher) sind PDL beschichtet Nacht und bereit für die Erstellung von Astrozyten Monoschichten. </li><li> Primäre Astrozyten Kultur ist für 5 Minuten mit 0,1% Trypsin Trypsin behandelt. Trypsin wird durch Zugabe von DMEM mit 10% FCS und 1% PSF und Zell am 300G für 5 Minuten zentrifugiert ergänzt inaktiviert. (Siehe Abbildung 2) </li><li> Astrozyten sind bei 1x10 5 Zellen / ml resuspendiert. 1 mL dieser Lösung ist es, jedem PDL beschichtet und in 4-Well-Platten aufgenommen. Astrozyten sind für 24-48 Stunden kultiviert, bis die Monoschicht vollständig konfluent. </li><li> Um Schwann-Zell-Migration auf Astrozyten Monoschichten zu beurteilen, parallel zur Schaffung Astrozyten Monoschichten, kreisförmigen PDL-beschichteten Deckgläschen in einer 50 ml-Röhrchen gegeben und zerkleinert mit einem Kunststoff-Pipette, um kleine Bruchstücke zu schaffen. </li><li> Glas-Fragmente werden in einen Brunnen in 6-Well-Platte überführt und mit einer Pinzette eignet große Fragmente ausgewählt und platziert in anderen Brunnen. 5 Glas-Fragmente werden in jedes Well in 6-Well-Platten gelegt. </li><li> Primäre Schwann-Zellen in Kolben kultiviert werden für 3 Minuten mit 0,1% Trypsin Trypsin behandelt. Trypsiniert Schwann-Zellen übertragen werden zu 15 ml Falcon-Röhrchen zu trennen und zentrifugiert bei 300 G für 5 Minuten. Schwann-Zellen sind mit 2x 10 6 Zellen / ml in DMEM/10% FCS / 1% PSF resuspendiert. </li><li> 20 uL Tropfen von Schwann-Zellen Suspension wird über je 5 Deckglas Fragmente in 6-Well-Platten und Zellen gelegt werden bei 37 ° C, 7% CO 2 für 2 Stunden inkubiert. </li><li> Nach 2 Stunden werden die Schwann-Zellen, die Bruchstücke Deckglas befestigt und DMEM/10% FCS / 1% PSF (ergänzt mit 2 um von Forskolin und 10 ug / mL der BPE ist zu jeder Vertiefung gegeben, so dass die Fragmente vollständig mit Medium bedeckt) . </li><li> Die 6-Well-Platten, die die Fragmente werden dann in den Inkubator für 24-48 Stunden gestellt, bis die Glassplitter vollständig confließend mit Schwann-Zellen. </li><li> Sobald Fragmente konfluent mit Schwann-Zellen sind, ist jedes Fragment vorsichtig mit scharfen Zange nahm und tauchte in HBSS auf ungebundene Zellen zu entfernen und dann umgedreht dem Gesicht nach unten auf Astrozyten Monoschicht. </li><li> Jedes der gut wird dann mit 1 ml DMEM/10% FCS / 1% PSF und Schwann-Zellen erlaubt, für 24 bis 48 Zeitraum migrieren abgedeckt sind. Während dieser Zeit experimentelle Reagenzien wie Wachstumsfaktoren zugesetzt werden, um deren Auswirkung auf die Anzahl und Abstand der Migration von Schwann-Zellen zu beurteilen. </li><li> Nach der Zeit der Völkerwanderung, sind die Glassplitter auf dem Boden jeder Vertiefung durch Zugabe von 4% Paraformaldehyd (PFA) für 30 Minuten fixiert. Nach Fixierung immunostain den Schwann-Zellen für p75 (Erkennen geringe Affinität NGF-Rezeptor), dann färben sie mit ABC Streptavidin Kit-Diaminobenzidin (DAB), um die Visualisierung der Schwann-Zellen unter dem Lichtmikroskop zu ermöglichen. </li></ol> 3. Repräsentative Ergebnisse: Nach 8-10 Tagen Schwann-Astrozyten Co-Kulturen zeigen eine scharfe Grenze zwischen den beiden Zelltypen. Diese Grenze ist stärker ausgeprägt, wenn Forskolin und BPE in der Kultur genutzt wird, wie Schwann-Zellen wird verbessert. Wenn keine Grenze während des Zeitraums von 10 Tagen beobachtet wird, kann die Zeit der Kultur weiter erhöht werden, bis die Grenzen festgelegt sind. Ändern des Verhältnisses der Zelldichte ist eine alternative Möglichkeit, die Grenze Bildung zu erhöhen. Ein Verhältnis von 3:1 Schwann: Astrozyten Zelldichte kann bei der Vorbereitung von Zellsuspensionen 8,18 sein. Nach Immunfärbung der Schwann-Zellen und Astrozyten zu identifizieren, zu Zelle Vermischung eine Linie gezogen werden, wo die Grenze ist festgelegt und die Zahl der Schwann-Zellen wandern in die Astrozyten Gebiet kann unter dem Fluoreszenzmikroskop gezählt werden zu beurteilen. Unten sind zwei Beispiele von Bildern erhalten Grenzen, eine mit gut ausgebildeten Trennung von Schwann-Zellen und Astrozyten (Abbildung 1A) und eine, wo die Zellen nicht getrennt haben und in verschiedene Gebiete aufgrund schlechter Technik (Abbildung 1B). <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Abbildung 1" /> . Abbildung 1 Boundary-Test: Schwann-Astrozyten-Interaktion. A) Eine 10 Tage Co-Kultur-Assay in Gegenwart von Forskolin und BPE zu Schwann-Zellen zu stimulieren zeigt eine scharfe Grenze zwischen den beiden Zelltypen (rot = P75, grün = GFAP). B) A 10 Tage Co-Kultur-Test, wo die Grenze nicht gebildet ist und die beiden Zellen Fronten gemischt. Dies war aufgrund der beiden Tröpfchen Mischen beim Platzieren Astrozyten und Schwann-Zellen Tröpfchen nebeneinander bei der Vorbereitung der Grenze (rot = P75, grün = GFAP). <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Abbildung 2" /> Abbildung 2. Representative Bild von konfluenten Astrozyten Monoschicht Migration-Assays beurteilen Bewegung eines Zelltyp über die Oberfläche eines anderen Zelltyp und somit prüfen, um ein anderes Phänomen aus, daß der Grenze Bildung. Praxis ist notwendig, um Umdrehen der Deckgläser auf Monoschichten sicher nicht zu einer Beschädigung der Schwann-Zell-bedeckten Glassplittern und Astrozyten Monoschichten durch die scharfen Zangen. Beschädigung Zur Beurteilung der Migration von Schwann-Zellen, die maximale Entfernung der Migration und die Anzahl der wandernden Zellen vom Rand des Deckglases kann unter dem Lichtmikroskop gemessen werden. Unten ist das Bild von Schwann-Zellen mit P75 Antikörper mit DAB-Färbung auf der Oberfläche der Astrozyten Monoschicht (Abbildung 3) gefärbt. <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Migration Assay mit invertierten Deckglas Assay. A) Schwann-Zellen (braun, immungefärbt mit p75-Antikörper) Migration weg von der Kante des Deckglases. Der Pfeil zeigt auf die Richtung der Zellmigration vom Deckglas.

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter.">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Schwann+cell+interactions+in+culture.">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li><li>Ghirnikar, R. S., Eng, L. 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Die oben beschriebenen Assays wurden in verschiedenen Studien, die die Rolle der verschiedenen Faktoren in Grenzen Bildung zwischen den Schwann-Astrozyten und begrenzt Schwann Zell-Migration in Astrozyten Umfeld eingebunden worden. Das Verständnis der Mechanismen, die diese Ereignisse ist wichtig, da es erlauben würde, die Entwicklung von Strategien zur Optimierung und Verbesserung der Integration von Schwann-Zellen Transplantate nach der Transplantation und damit erleichtern Sie die Ausfa...

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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>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

Schwann-Zellen sind eine der häufigsten verwendete in Reparatur befindlichen Zellen Strategien nach Rückenmarksverletzungen. Schwann-Zellen sind in der Lage zu unterstützen axonale Regeneration und Sprossung durch Sekretion Wachstumsfaktoren 1,2 und Bereitstellung von wachstumsfördernden Adhäsionsmoleküle 3 und extrazelluläre Matrix Moleküle 4. Außerdem sind sie myelinisieren der demyelinisierte Axone an der Stelle der Verletzung 5. Doch nach der Transplantation, tun Schwann-Zellen nicht von der Stelle des Implantats zu migrieren und nicht mit dem Host-Astrozyten 6,7 vermischen. Dies führt zur Bildung einer scharfen Grenze zwischen Schwann-Zellen und Astrozyten, wodurch ein Hindernis für den Anbau von Axonen versucht, das Transplantat wieder in das Wirtsgewebe proximal und distal zu verlassen. Astrozyten in Kontakt mit Schwann-Zellen auch zu unterziehen Hypertrophie und Up-Regulierung der hemmenden Moleküle 8-13. In vitro-Untersuchungen haben zu modellieren Schwann-Zell-Interaktionen Astrozyten verwendet worden und wurden in das Verständnis des Mechanismus der zellulären Verhaltens wichtig. Diese in-vitro-Assays gehören Grenze Assay, wo eine Co-Kultur unter Verwendung von zwei verschiedenen Zellen, die mit jedem Zelltyp zu besetzen verschiedene Gebiete mit nur ein kleiner Spalt zwischen den beiden Fronten Zelle ist. Da die Zellen teilen sich und wandern, bekommen die beiden zellulären Fronten einander näher und schließlich kollidieren. Dies ermöglicht dem Verhalten der beiden Zellpopulationen an der Grenze analysiert werden. Eine weitere Variante der gleichen Technik ist es, die beiden Zellpopulationen in der Kultur-Mix und im Laufe der Zeit die beiden Zelltypen mit Schwann-Zellen zu trennen verklumpten als Inseln zwischen Astrozyten zusammen Erstellen mehrerer Schwann-Astrozyten Grenzen. Der zweite Test in der Untersuchung der Interaktion von zwei Zelltypen ist die Migration-Assay, wo zelluläre Bewegung auf der Oberfläche der anderen Zelltyp Monoschicht 14,15 verfolgt werden können. Dieser Test wird im Allgemeinen als umgekehrt Deckglas Test bekannt. Schwann-Zellen werden auf kleine Glassplitter kultiviert und sie sind Gesicht invertiert hinunter auf die Oberfläche der Astrozyten Monoschichten und Migration ist von der Kante des Deckglases beurteilt. Beide Tests wurden bei der Untersuchung der zugrunde liegenden Mechanismen der zellulären Ausgrenzung und Grenzen Bildung beteiligt instrumental. Einige der identifizierten Moleküle mit diesen Techniken gehören N-Cadherine 15, Chondroitinsulfat Proteoglykane (CSPGs) 16,17, FGF / Heparin 18, Eph / Ephrine 19. Dieser Artikel will Grenze Assay und Migration-Assay in schrittweise beschreiben und erläutern die möglichen technischen Probleme, die auftreten könnten.

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Dieser Artikel will in schrittweise beschreiben die gebräuchlichsten<em&gt; In-vitro-</em&gt; Tests in Studium Schwann-Zell-Interaktionen asrtocyte verwendet.

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 ...

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Research

JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.2K Aufrufe.  University of Cambridge. Dieser Artikel will in schrittweise beschreiben die gebräuchlichsten In-vitro- Tests in Studium Schwann-Zell-Interaktionen asrtocyte verwendet.

Serie: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Schnelle Homogene Detektion von biologischen Assays mit magnetischen Modulation Biosensorik-System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Forschung

Biologie

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

In-vitro-Analyse der PDZ-abhängigen CFTR Makromolekulare Signalkomplexe

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

In-vitro-Zellmigration und Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Die Analyse der RNA-Prozessierung Reaktionen mit Handy Free Systems: 3 &#39;Ende Spaltung von prä-mRNA-Substrate<em&gt; In-vitro-</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

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 ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Vergleichende Analyse von Human Growth Hormone im Serum Mit SPRi, Nano-SPRi und ELISA-Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Wirkung von fluoreszierenden Proteinen auf Fusion Partner mit Trinukleotiderkrankungen Toxizität Assays in Hefe

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Mikrobiota-Analyse mit zweistufiger PCR- und 16S-rRNA-Gensequenzierung der nächsten Generation

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analyse der E3-Ubiquitin-Ligase-Funktion

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as regulators of various biological processes. Notably, emerging evidence demonstrates that host circRNAs can be differentially expressed (DE) upon infection with&#160;pathogens (e.g., influenza and coronaviruses), suggesting a role for circRNAs in regulating host innate immune responses. However, investigations on the role of circRNAs during pathogenic infections are limited by the knowledge and skills required to carry out the necessary bioinformatic analysis to identify DE circRNAs from RNA sequencing (RNA-seq) data. Bioinformatics prediction and identification of circRNAs is crucial before any verification, and functional studies using costly and time-consuming wet-lab techniques. To solve this issue, a step-by-step protocol of in silico prediction and characterization of circRNAs using RNA-seq data is provided in this manuscript. The protocol can be divided into four steps: 1) Prediction and quantification of DE circRNAs via the CIRIquant pipeline; 2) Annotation via circBase and characterization of DE circRNAs; 3) CircRNA-miRNA interaction prediction through Circr pipeline; 4) functional enrichment analysis of circRNA parental genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). This pipeline will be useful in driving future in vitro and in vivo research to further unravel the role of circRNAs in host-pathogen interactions.

In Silico Identification and Characterization of circRNAs During Host-Pathogen Interactions

The protocol submitted here explains the complete in silico pipeline needed to predict and functionally characterize circRNAs from RNA-sequencing transcriptome data studying host-pathogen interactions.

In Silico Identifizierung und Charakterisierung von circRNAs während Wirt-Pathogen-Interaktionen

Circular RNAs (circRNAs) are a class of non-coding RNAs that are formed via back-splicing. These circRNAs are predominantly studied for their roles as ...

Methods Article

Analyse von Schwann-Astrozyten Wechselwirkungen mit In Vitro Assays