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

1 Préparation des cellules (J-2 à 0) <ol><li> Fractionner les cellules HEK293 1: 5 en utilisant 0,05% de solution de trypsine-EDTA et incuber à la croissance des médias à 37 ° C avec 5% de CO 2 jusqu&#39;à 60 ans - 80% de confluence (2 - 3 jours). Pour chaque condition, les cellules en culture dans 2 x boîtes de 100 mm. </li></ol> 2. transfection des cellules (Jour 1) <ol><li> Transfecter des cellules HEK293 avec le plasmide codant pour la Fc-fusion en utilisant un réactif de transfection, tels que la lipofectamine. D&#39;autres méthodes qui conduisent à des efficacités de transfection comparables peuvent également être utilisés. </li><li> Retour cellules transfectées à l&#39;incubateur pendant 24 heures. </li></ol> 3. cellulaire propagation (Jour 2) <ol><li> Préchauffer milieux de croissance sans sérum bovin fœtal (FBS) à 37 ° C. </li><li> Rincer la vaisselle 2x avec 10 ml de croissance médias FBS, et renvoient les plats à la 37 ° C incubateur pendant 1 heure. </li><li> Rincer la cultures des plats une fois de plus avec 10 ml de croissance médias FBS, pour un total de 3 lavages. </li><li> Incubate les cellules HEK 293 transfectées à 37 ° C pendant 48 heures sans autre FBS avant de recueillir médias. </li></ol> 4 perles d&#39;agrégation (Jour 4) <ol><li> Pour recueillir des médias de boîtes de culture, de transférer les médias de chaque paire de plats à 50 ml tubes coniques et rotation à 500 g pendant 5 min pour sédimenter les débris cellulaires. </li><li> Les milieux filtrants de 50 ml tube conique dans un filtre centrifuge utilisant une seringue de 30 ml et de 0,45 um filtre de la seringue. </li><li> Spin filtres centrifuges à 4000 x g et 4 ° C jusqu&#39;à ce que le volume de milieu de culture concentré est d&#39;environ 500 pl (environ 15 min). Répétez jusqu&#39;à ce que tous les supports de culture a été ajouté et concentré. </li><li> Ajouter 1,5 ul de billes magnétiques protéines G à 1 ml de glace froide Binding Buffer dans un tube de 1,5 ml pour chaque échantillon, les placer sur un aimant et éliminer le tampon. Immédiatement ajouter au milieu de culture concentré de la protéine G des billes magnétiques. </li><li> Faire tourner les tubes à 4 ° C pendant 2 heures. </li><li> Pltubes ace sur un aimant et supprimer médias. Laver rapidement perles deux fois avec le froid de la glace 1 ml Binding Buffer, puis remettre en suspension des billes dans 300 pi Binding Buffer. </li><li> De Split remis en suspension dans 2 perles tubes, 150 pi dans chaque tube, puis ajouter 1,5 pi de mM CaCl 2 ou 200 200 mM EDTA pour le &quot;calcium&quot; et les conditions &quot;pas de calcium&quot;, respectivement. </li><li> Transfert 100 pi de chaque condition à une dépression bien glisser et de recueillir des micrographies au microscope lumière transmise (figure 2). Recueillir des images à partir de 5 champs de vision pour chaque expérience à chaque point de temps souhaité. </li></ol> Analyse 5. données <ol><li> ImageJ, ou logiciel d&#39;analyse d&#39;image comparable, ouvrez l&#39;un des cinq ensembles de données d&#39;image en utilisant le / séquence d&#39;images Importer ... dans le menu déroulant Fichier. Ceux-ci seront ouverts comme une pile d&#39;images. </li><li> Dans la boîte de dialogue ... image / Propriétés, modifiez la iunités de mage à pixels et mis Largeur Pixel et Hauteur de pixel à 1,0. </li><li> Convertir les images en binaire utilisant le / Seuil Ajustez ... de commande dans le menu déroulant Image. Régler le seuil pour inclure pixels qui contribuent à des billes ou des agrégats de perles, mais qui excluent fond et de petites particules. Appliquer à toutes les images de la pile. </li><li> Dans la boîte de dialogue de mesures Set, cochez les cases de la région et la pile de position. </li><li> Exécutez analyser des particules ... dans le menu déroulant Analyser. Cela va générer une liste de particules identifiées, notamment leur taille (région) et l&#39;image dans laquelle ils ont été identifiés. </li><li> Répétez ce processus pour que l&#39;expérience et l&#39;état expérimental. </li></ol>

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The authors declare that they have no competing financial interests.

L&#39;adhésion cellulaire est un élément essentiel de la vie multicellulaire et est médiée par une large gamme de protéines de la surface cellulaire. Parmi ceux-ci, les propriétés adhésives détaillées sont comprises que pour une part relativement faible. Ici, nous avons décrit un protocole simple et rapide pour enquêter sur la capacité adhésive homophile de ectodomaines sécrétées fusionnées à une étiquette d&#39;épitope pratique. Cette approche a un certain nombre d&#39;avantages importants. Premi?...

L&#39;adhérence cellule-cellule est essentielle pour le développement et l&#39;intégrité des organismes multicellulaires et est médiée par une grande diversité de molécules de la surface cellulaire. Beaucoup de ces molécules d&#39;adhésion ont été identifiés et caractérisés, mais beaucoup restent à découvrir. Plusieurs méthodes sont utilisées pour étudier les propriétés des molécules d&#39;adhésion cellulaire (CAM), y compris le tri cellulaires essais, des tests d&#39;agrégation de cellules 1-8 et méthodes biophysiques, comme la microscopie à force atomique et la surface spectroscopie de force 9-12. La complexité de la même simplifié dans des systèmes in vitro utilisant des lignées cellulaires, il est difficile de déterminer les propriétés adhésives d&#39;une CAM putatif. Typiquement, une molécule est considérée comme une CAM si elle induit une agrégation cellulaire lorsqu&#39;il est transfecté dans une lignée cellulaire non-adhésif. Toutefois, il est clair qu&#39;il ne s&#39;agit pas de preuve directe de l&#39;activité de l&#39;adhésif. Par exemple, faciliter la distribution de surface de la cellule ou de la stabilité d&#39;unCAM se traduirait également par l&#39;agrégation cellulaire accrue 1,13. De plus, un vrai CAM peut échouer la médiation agrégation cellulaire si la lignée cellulaire n&#39;a pas d&#39;autres co-facteurs nécessaires pour la livraison de la surface cellulaire ou de stabilisation. Pour éviter ces facteurs de risque, des tests plus directs peuvent être employées qui sont fondées sur l&#39;idée que les interactions adhésives doivent être une propriété intrinsèque biochimique du domaine extracellulaire. Alors que les perles étaient initialement utilisés pour caractériser Ng-CAM 2, ces essais ont été étendus afin d&#39;enquêter sur la cadhérine-médiation adhérence 12,14,15. Utilisation de fusion des fusions C-cadhérine ectodomaine-Fc, le laboratoire a montré que Gumbiner multiples répétitions de cadhérine contribuent à des interactions homophiles 14. En utilisant des tests d&#39;agrégation de perles comparables, la E-cadhérine et la N-cadhérine adhésion ont également été caractérisé 12,15, comme l&#39;ont fait un certain nombre de PROTOCADHERINES 1,15-18 et isoformes DSCAM de Drosophila <em&gt; 19. Nous décrivons ici un test relativement simple et rapide pour la caractérisation de l&#39;activité adhésive de sécrétées, ectodomaines de marquage épitopique de cames de homophiles putatifs (figure 1). Nous avons utilisé ce test essentiellement de caractériser les membres de la superfamille des cadhérines.

<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

L&#39;adhérence cellule-cellule est essentielle à la vie multicellulaire et est médiée par une grande diversité de protéines de la surface cellulaire. Cependant, les interactions adhésives pour un grand nombre de ces protéines sont mal compris. Nous présentons ici une méthode simple et rapide pour caractériser les propriétés adhésives des homophiles des molécules d&#39;adhésion cellulaire putatifs. De culture des cellules HEK293 sont transfectées avec l&#39;ADN plasmidique codant pour une sécrétion, un ectodomaine d&#39;une étiquette d&#39;épitope d&#39;une protéine de surface cellulaire. Utilisation de billes fonctionnalisées spécifiques pour le marqueur d&#39;epitope, le soluble, la protéine de fusion sécrétée est capturé à partir du milieu de culture. Les billes enrobées peuvent alors être utilisés directement dans des tests d&#39;agrégation billes ou à bille fluorescente tri dosages pour tester l&#39;adhérence homophile. Si on le souhaite, la mutagenèse peut être utilisée pour élucider les acides aminés ou domaines spécifiques nécessaires pour l&#39;adhérence. Ce dosage ne nécessite que de petites quantités de protéine exprimée, ne nécessite pas la production de lignées cellulaires stables, et peut être accomplished à 4 jours.

<html> Une expérience d&#39;exemple est présenté sur la figure 2, qui représente l&#39;agrégation des billes de calcium-dépendante par l&#39;ectodomaine de la N-cadhérine fusionné à Fc (NcadEC-Fc). En l&#39;absence de calcium, les perles présentent peu ou pas de tendance à l&#39;agrégation et il n&#39;y a pas d&#39;augmentation de la taille des agrégats avec le temps (Figure 1A, C). En présence de calcium, des perles revêtues d&#39;NcadEC-Fc montrent une agrégation solide...

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.

Perles agrégation essais pour la caractérisation de molécules d&#39;adhésion cellulaire putatif

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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Unit 1

Cell-Cell Interactions

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10.4K vues.  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.

Vidéo: 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).

Synthèse, l&#39;Assemblée, et caractérisation des nanoparticules Monocouche protégées Films d&#39;or pour Electrochimie monocouche de protéines

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

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Bio-ingénierie

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.

Essai Fréquence d&#39;adhérence pour In Situ Analyse cinétique de la Croix-jonctionnelle interactions moléculaires à l&#39;interface cellule-cellule

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.

Utilisant une impédance Cell-substrat et imagerie de cellules vivantes pour mesurer les changements en temps réel dans adhésion cellulaire et De-adhérence induites par 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 ...

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.

Environnement microfluidique contrôlée pour Dynamic Investigation of Blood Red Cell Agrégation

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.

Wet Chemistry and Peptide Immobilisation sur polytétrafluoroéthylène pour l&#39;amélioration de l&#39;adhérence cellulaire

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.

L&#39;agrégation magnétique de cellules souches 3D et bioréacteurs Maturation pour Cartilage régénération

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.

Développement de dispositifs microfluidiques pour étudier la capacité d’élongation de la pointe de croissance des cellules végétales dans des espaces très réduits

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.

Détection d’endotoxines dans les Nano-formulations utilisant la Limulus Amébocytes Lysate (LAL)

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.

Une plate-forme microfluidique multicouche pour la conduite d'une expression génique prolongée sans cellules

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.

Fabrication d’une nanomatrice biomimétique avec nanotubes de base Janus et fibronectine pour l’adhérence aux cellules souches

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