This work was supported by an NSF/ARRA award (IOS 0920357) and an award from the NIH (5R21MH098463).

1. Zellpräparation (Tag -2 bis 0) <ol><li> Split HEK293-Zellen 1: 5 unter Verwendung von 0,05% Trypsin-EDTA-Lösung und Inkubation in Wachstumsmedium bei 37 ° C mit 5% CO 2, bis 60 bis 80% konfluent (2 - 3 Tage). Für jede Bedingung Kulturzellen in 2 x 100-mm-Schalen. </li></ol> 2. Zell Transfektion (Tag 1) <ol><li> Transfektion von HEK293-Zellen mit Plasmid-Codierung Fc-Fusion mit einem Transfektionsreagenz Lipofectamine wie. Alternative Methoden, die in vergleichbaren Transfektionseffizienzen führen, können ebenfalls verwendet werden. </li><li> Rück transfizierten Zellen für 24 Stunden Inkubator. </li></ol> 3. Zellvermehrung (Tag 2) <ol><li> Vorheiz Growth Medium ohne fötales Rinderserum (-FBS) bis 37 ° C beträgt. </li><li> Spülen Geschirr 2x mit 10 ml Wachstumsmedien -FBS, und geben die Gerichte an die 37 ° C-Inkubator für 1 Stunde. </li><li> Spülen der Kulturen Geschirr noch einmal mit 10 ml Wachstumsmedien -FBS, für insgesamt 3 Waschungen. </li><li> IncuBeize die transfizierten HEK293-Zellen bei 37 ° C für weitere 48 h ohne FBS vor dem Sammeln Medien. </li></ol> 4. Bead Aggregation (Tag 4) <ol><li> Medien von Kulturschalen sammeln, übertragen Sie Medien von jedem Paar von Gerichten zu 50 ml konischen Röhrchen und Spin bei 500 g für 5 min, um Zelltrümmer zu pelletieren. </li><li> Filtermedien 50 ml konischen Röhrchen in ein Zentrifugalfilter mit einer 30 ml Spritze und 0,45 um-Spritzenfilter. </li><li> Spin Zentrifugalfilter bei 4.000 × g und 4 ° C bis zum Volumen der konzentrierten Kulturmedium beträgt etwa 500 ul (etwa 15 min). Wiederholen, bis alle Kulturmedien wurde hinzugefügt und eingeengt. </li><li> Fügen 1,5 ul Protein G magnetischen Kügelchen auf 1 ml eiskaltem Bindungspuffer in einem 1,5 ml Mikrozentrifugenröhrchen für jede Probe, auf ein Magnet und entfernen Puffer. Sofort die geballte Kulturmedien, um das Protein G magnetischen Beads hinzuzufügen. </li><li> Drehen Röhrchen bei 4 ° C für 2 Stunden. </li><li> PlAss Rohre auf einem Magneten und Medien zu entfernen. Waschen schnell Perlen zweimal mit eiskaltem 1 ml Binding Buffer, und dann wieder zu suspendieren Perlen in 300 ul Binding Buffer. </li><li> Split resuspendiert Perlen in 2 Röhren, 150 ul in jedes Rohr, und fügen Sie 1,5 ul 200 mM CaCl 2 oder 200 mM EDTA für die &quot;Calcium&quot; und &quot;kein Kalzium&quot; Bedingungen auf. </li><li> Transfer 100 ul von jedem Zustand in einen Depression gut schieben und sammeln mikroskopischen Aufnahmen mit einem Durchlichtmikroskop (Abbildung 2). Sammeln Sie Bilder von 5 Sichtfelder für jedes Experiment an jedem gewünschten Zeitpunkt. </li></ol> 5. Datenanalyse <ol><li> Mit ImageJ oder vergleichbare Bildanalyse-Software, öffnen Sie eine der fünf Bilddatensätze mit dem Import / Bildsequenz ... aus dem Pulldown-Menü Datei. Diese werden als Bildstapels geöffnet werden. </li><li> Im Dialogfeld Bild ... / Eigenschaften, ändern Sie die iMagier Einheiten auf Pixel und setzen Pixel Breite und Höhe bis 1,0 Pixel. </li><li> Konvertieren Sie die Bilder in dual mit dem Anpassen / Schwellenwert ...-Befehl in der Bild Pulldown-Menü. Stellen Sie den Schwellenwert auf Pixel, die Perlen oder Perlen Aggregate beitragen, umfassen, aber das auszuschließen Hintergrund und kleinen Partikeln. Gelten für alle Bilder im Stapel. </li><li> Im Dialogfeld Set Maße, überprüfen Sie die Umgebung und Stack Position Boxen. </li><li> Analyze Particles ... in der Analyse Pulldown-Menü. Dadurch wird eine Liste der identifizierten Partikel, einschließlich ihrer Größe (Fläche) und das Bild in der sie erkannt zu generieren. </li><li> Wiederholen Sie diesen Vorgang für die Experimente und Versuchsbedingung. </li></ol>

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J., Jontes, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+complex+of+Protocadherin-19+and+N-cadherin+mediates+a+novel+mechanism+of+cell+adhesion.">A complex of Protocadherin-19 and N-cadherin mediates a novel mechanism of cell adhesion.</a> J. Cell Biol. 195 (7), 1115-1121 (2011).</li><li>Biswas, S., Emond, M. R., Jontes, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clustered+protocadherins+Pcdhalpha+and+Pcdhgamma+form+a+heteromeric+complex+in+zebrafish.">The clustered protocadherins Pcdhalpha and Pcdhgamma form a heteromeric complex in zebrafish.</a> Neuroscience. 219, 280-289 (2012).</li><li>Blevins, C. J., Emond, M. R., Biswas, S., Jontes, J. 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The authors declare that they have no competing financial interests.

Zelladhäsion ist ein wesentliches Merkmal der vielzelligen Leben und wird von einem breiten Spektrum von Zelloberflächenproteinen vermittelt. Von diesen sind die detaillierten Klebeeigenschaften nur für einen relativ kleinen Anteil verstanden. Hier haben wir ein einfaches, schnelles Protokoll zur Untersuchung der homophile Haftfähigkeit von sekretierten Ektodomänen in einem geeigneten Epitop-Tag fusioniert beschrieben. Dieser Ansatz hat eine Reihe von wichtigen Vorteilen. Zuerst werden die stabilen Zelllinien nicht...

Zell-Zell-Adhäsion ist wesentlich für die Entwicklung und die Integrität von mehrzelligen Organismen und durch eine Vielfalt von Zelloberflächenmolekülen vermittelt. Viele dieser Adhäsionsmoleküle wurden identifiziert und charakterisiert, obwohl viele noch zu entdecken sind. Verschiedene Methoden werden verwendet, um die Eigenschaften von Zelladhäsionsmolekülen (CAMs), einschließlich Zellsortierung Assays, Zellaggregationsassays 1-8 und biophysikalische Methoden, wie die Rasterkraftmikroskopie und Oberflächenkraftspektroskopie 9-12 untersuchen. Die Komplexität auch in vitro-Systemen unter Verwendung von Zelllinien vereinfacht macht es schwierig, die Hafteigenschaften eines mutmaßlichen CAM bestimmen. Typischerweise wird ein Molekül als ein CAM, wenn die Zellaggregation induziert, wenn sie in einem nicht-klebenden Zelllinie transfiziert. Es ist jedoch klar, dass dies keine direkte Hinweise auf Haftungsaktivität. Beispielsweise erleichtert die Zelloberfläche Lieferung oder Stabilität einesCAM würde auch zu einer erhöhten Zellaggregation 1,13 führen. Darüber hinaus kann eine wahre CAM nicht, Zellaggregation vermitteln, wenn die Zelllinie fehlt anderen Co-Faktoren für Zelloberflächen Lieferung oder Stabilisierung erforderlich. Um diese erschwerenden Faktoren zu vermeiden, mehr direkte Assays eingesetzt werden, die auf der Idee basieren, dass adhäsive Wechselwirkungen sollte eine intrinsische biochemische Eigenschaft der extrazellulären Domäne sein. Während Perlen wurden zunächst zur Ng-CAM 2 zu charakterisieren, haben diese Tests, um Cadherin-vermittelte Adhäsion 12,14,15 untersuchen erweitert. Verwendung Fusion des C-Cadherin-Ektodomäne-Fc-Fusionen zeigten die Gumbiner Labor dass mehrere Cadherin Wiederholungen tragen zu Interaktionen 14 homo. Mit vergleichbaren Wulst Aggregationstests, E-Cadherin und N-Cadherin-Haft wurden ebenfalls gekennzeichnet 12,15, als eine Reihe von Protocadherinen 1,15-18 und Dscam Isoformen von Drosophila haben <em&gt; 19. Hier beschreiben wir einen relativ einfachen und schnellen Test zur Charakterisierung der Klebstoff Aktivität sezerniert Epitop markierte Ektodomäne von mutmaßlichen homo Nocken (Abbildung 1). Wir haben diesen Test verwendet hauptsächlich die Mitglieder der Cadherin-Oberfamilie charakterisieren.

<table border="0" cellpadding="0" cellspacing="0" width="630">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:216px;">Company</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">pFc-N1 plasmid</td>
			<td style="width:203px;">To be submitted</td>
			<td style="width:216px;">Addgene</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">HEK293 cells</td>
			<td style="width:203px;">CRL-1573</td>
			<td style="width:216px;">American Type Culture Collection</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">DMEM</td>
			<td style="width:203px;">10-013-CV</td>
			<td style="width:216px;">Mediatech - Corning</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">F2442</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">100X Pen-Strep (10,000 U/ml)</td>
			<td style="width:203px;">15140-122</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">0.05% Trypsin-EDTA</td>
			<td style="width:203px;">25300054</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Lipofectamine 2000</td>
			<td style="width:203px;">11668030</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Dynabeads &#8211; Protein G</td>
			<td style="width:203px;">10003D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:212px;">Bovine Serum Albumin</td>
			<td style="width:203px;">A3294</td>
			<td style="width:216px;">Sigma</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Depression slides</td>
			<td style="width:203px;">71878-06</td>
			<td style="width:216px;">Electron Microscopy Sciences</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:212px;">Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane</td>
			<td rowspan="2" style="width:203px;">UFC901024</td>
			<td rowspan="2" style="width:216px;">Millipore</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">0.45mm syringe filter</td>
			<td style="width:203px;">83.1826</td>
			<td style="width:216px;">Sarstedt</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Dynamag-2 Magnet</td>
			<td>12321D</td>
			<td style="width:216px;">Life Technologies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:212px;">Fiji (ImageJ) Image Analysis Software</td>
			<td style="width:203px;"></td>
			<td style="width:216px;">http://fiji.sc/Fiji</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;">Table of Buffers/Solutions</td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:212px;"></td>
			<td style="width:203px;"></td>
			<td style="width:216px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media</td>
			<td style="width:203px;">DMEM + 10% FBS + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:212px;">Growth Media &#8211; FBS</td>
			<td style="width:203px;">DMEM + Pen-Strep</td>
			<td style="width:216px;">Filter sterilize and store at 4&#176;C.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:212px;">Binding Buffer</td>
			<td style="width:203px;">50 mM Tris, pH 7.4, 100 mM NaCl, 10 mM KCl, 0.2% BSA</td>
			<td style="width:216px;">Vortex until dissolved, keep on ice.</td>
		</tr>
	</tbody>
</table>

bead aggregation assays for the characterization of putative cell adhesion molecules

Zell-Zell-Adhäsion ist von grundlegender Bedeutung für Vielzeller und wird durch eine Vielfalt von Zelloberflächenproteinen vermittelt. Jedoch sind die adhäsiven Wechselwirkungen vieler dieser Proteine ​​schlecht verstanden. Hier präsentieren wir Ihnen eine einfache, schnelle Methode zur Charakterisierung der Hafteigenschaften von mutmaßlichen homophile Zell-Adhäsionsmoleküle. Kultivierten HEK293-Zellen mit Plasmid-DNA, kodierend eine sekretierte, Epitop-tagged Ektodomäne eines Zelloberflächenprotein transfiziert. Unter Verwendung von für die Epitop-Markierung funktionalisierten Kügelchen wird das lösliche, sekretierte Fusionsprotein aus dem Kulturmedium festgehalten. Die beschichteten Perlen können dann direkt in Perlenaggregationstests oder fluoreszierende Perle Sortier Assays für homophile Adhäsion zu prüfen. Falls gewünscht, kann die Mutagenese dann verwendet, um die spezifische Aminosäuren oder Domänen für die Haftung erforderlichen aufzuklären. Dieser Test erfordert nur geringe Mengen an exprimiertem Protein, nicht die Herstellung von stabilen Zelllinien erfordern, und kann acco werdeninnerhalb von 4 Tagen mplished.

<html> Ein Beispiel ist in Abbildung 2, die Kalzium-abhängige Aggregation zeigt Wulst von der Ektodomäne von N-Cadherin-Fc (NcadEC-Fc) fusioniert dargestellt. In Abwesenheit von Calcium, Perlen weisen eine geringe oder keine Tendenz zu aggregieren, und es gibt keine Zunahme der Gesamtgröße mit der Zeit (1A, C). In Anwesenheit von Calcium, Perlen mit NcadEC-Fc-Show robust Aggregation beschichtet, mit Aggregatgröße über die Zeit (Abbildung 1B, C) ​​zu. Dieses Ex...

Watch this Scientific Journal Video about Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules at JoVE.com

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Wulst Aggregationsassays zur Charakterisierung von putativen Zelladhäsionsmoleküle

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

bead-aggregation-assays-for-characterization-putative-cell-adhesion

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JoVE Journal

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Cell Biology

Bioengineering

Unit 1

Cell-Cell Interactions

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10.4K Aufrufe.  Ohio State University. Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Serie: Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Synthesis, Assembly und Charakterisierung von Monolayer Protected Gold-Nanopartikeln Films für die Protein-Monoschicht Elektrochemie

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Forschung

Biotechnik

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

Adhesion Frequency-Assay für In Situ Kinetics Analyse der Cross-Junktionaler Molecular Interactions an der Zell-Zell-Schnittstelle

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Benutzen Sie die Cell-Substrat-Impedanz und Live Cell Imaging, um Echtzeit-Änderungen in der Zelladhäsion und De-Haftung durch Matrix Änderung Induzierte Messen

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Controlled Environment Mikrofluidik für dynamische Untersuchung der Aggregation roter Blutzellen

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Nasschemie und Peptid Immobilisation auf Polytetrafluoroethylen für verbesserte Zellhaftung

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Magnetic Stem Cell Aggregation und Bioreactor Ausreifung für Knorpelregeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior in such restrictive conditions. To address this issue, we have developed growth chambers for tip-growing plant cells that contain a series of narrow, micro-fabricated gaps (~1 &#181;m) in a poly-dimethylsiloxane (PDMS) substrate. This transparent material allows the user to monitor tip elongation processes in individual cells during microgap penetration by time-lapse imaging. Using this experimental platform, we observed morphological changes in pollen tubes as they penetrated the microgap. We captured the dynamic changes in the shape of a fluorescently labeled vegetative nucleus and sperm cells in a pollen tube during this process. Furthermore, we demonstrated the capability of root hairs and moss protonemata to penetrate the 1 &#181;m gap. This in vitro platform can be used to study how individual cells respond to physically constrained spaces and may provide insights into tip-growth mechanisms.

We describe a method to investigate the capability of tip-growing plant cells, including pollen tubes, root hairs, and moss protonemata, to elongate through extremely narrow gaps (~1 &#181;m) in a microfluidic device.

Entwicklung von mikrofluidischen Geräten um die Dehnung Fähigkeit der Spitze wachsenden Pflanzenzellen in sehr kleinen Räumen zu studieren

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Eine multilayer mikrofluidische Plattform für die Leitung einer längeren zellfreien Genexpression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

Herstellung einer biomimetischen Nano-Matrix mit Janus Base Nanotubes und Fibronectin zur Stammzellhaftung

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...