Nous tenons à remercier Philippe Warren pour son aide genre.

1. Assay limites: <ol><li> Préparation de base: diapositives Chambre sont recouvertes de poly-D-Lysine lames de verre la nuit et stériles sont préparés avant de commencer l&#39;expérience. Milieu est préparé en utilisant un milieu Eagle modifié par Dulbecco (DMEM), supplémenté avec 10% sérum de veau foetal (FCS), et 1% de pénicilline-streptomycine-Fungizon (FSP). Aussi une bouteille de calcium / magnésium libre solution saline équilibrée de Hanks (HBSS) est préparé. </li><li> Primaire des cellules de Schwann de rat en culture dans le flacon sont trypsinisées pendant 3 minutes en utilisant 0,1% de trypsine. Primaire de la culture des astrocytes rat est trypsinisées pendant 5 minutes en utilisant 0,1% de trypsine. La trypsine est inactivé par l&#39;ajout de DMEM supplémenté avec 10% de SVF et 1% PSF. </li><li> Trypsinisées cellules de Schwann et les astrocytes sont transférés à séparer 15 ml tubes Falcon et centrifugés à 300G pendant 5 minutes. </li><li> Les cellules de Schwann et les astrocytes sont remis en suspension à la densité de 2 x 10 6 (voir la section des résultats représentatifs des variations de densité cellulaire). </li><li> 50 déposer uL de suspension de cellules de Schwann est placé à une extrémité du PDL puits recouverts de glisser chambre. Une bande de verre a été utilisé pour salir la chute vers le centre de la chambre de générer un bord droit. (Utilisation d&#39;un coupe-verre, un des lamelles de verre rectangulaires sont coupées longitudinalement afin qu&#39;il s&#39;adapte à la largeur de la chambre de diapositives utilisées pour des essais de la frontière) </li><li> Une deuxième baisse de 50 pi de la suspension des astrocytes a été placé à l&#39;extrémité opposée du même puits et barbouillé vers le centre pour créer une arête droite parallèle à la première goutte frottis avec un petit écart de 0,2 mm entre eux. </li><li> La chambre de diapositives contenant les gouttes sont placées dans l&#39;incubateur à 37 ° C pendant 2 heures. </li><li> Après 2 heures, les cultures ont été lavées avec du DMEM pour enlever non-inscrits débris cellulaires et du milieu frais contenant du DMEM / FCS à 10% / 1% PSF ajouté. Remarque: DMEM / FCS à 10% / 1% PSF peut être complétée par la forskoline (2 pm) et extrait pituitaire bovin (BPE) (10 pg / mL) stimulent la prolifération de cellules de Schwann. BPE fournir les facteurs de croissance nécessaires pour permettre à long terme de culture de cellules de Schwann et la forskoline augmente le taux adénosine monophosphate cyclique dans les cellules de Schwann à induire la prolifération </li><li> Les cellules sont cultivées pendant environ 8-10 jours à 37 ° C, 7% de CO 2 incubateur. Le milieu nécessite d&#39;être changée tous les 3 jours. Les deux fronts de cellule finira par atteindre l&#39;autre et d&#39;une limite nette se forme entre les deux types cellulaires. A ce stade, les expériences peuvent être réalisées analyser les facteurs impliqués dans la formation de frontière. </li><li> Co-culture peut être fixé à l&#39;aide de formaldéhyde 4% et les cellules de Schwann peuvent être immunocolorés avec des anti-P75 anticorps et les astrocytes avec des anticorps anti-GFAP pour identifier les cellules de co-culture. P75 anticorps reconnaît peu d&#39;affinité sur des récepteurs du NGF cellules de Schwann qui astrocytes manque. Anti-GFAP anticorps reconnaît gliales firbrillary protéine acide qui localise au cytoplasme d&#39;astrocytes. (S&#39;il vous plaît voir la Figure 1A pour la limite de son représentant) </li></ol> 2. Essai de migration: <ol><li> 4 assiettes bien (15mm puits) sont PDL couché la nuit et préparées pour la création de monocouches d&#39;astrocytes. </li><li> Primaire de la culture des astrocytes sont trypsinisées pendant 5 minutes en utilisant 0,1% de trypsine. La trypsine est inactivée par l&#39;ajout de DMEM supplémenté avec 10% de SVF et 1% de cellules PSF et centrifugés à 300G pendant 5 minutes. (Voir Figure 2) </li><li> Les astrocytes sont re-suspendue à 1x10 5 cellules / ml. 1 ml de cette solution est ajoutée à chaque revêtement PDL bien dans 4 assiettes bien. Les astrocytes sont cultivés pendant 24-48 heures jusqu&#39;à ce que la monocouche est complètement confluentes. </li><li> Afin d&#39;évaluer la migration des cellules de Schwann sur des monocouches d&#39;astrocytes, parallèlement à la création de monocouches d&#39;astrocytes, circulaire lamelles de verre enduit de PDL sont placés dans un tube de 50 ml et écrasés à l&#39;aide d&#39;une pipette en plastique pour créer de petits fragments de verre. </li><li> Des fragments de verre sont transférés dans un puits dans 6 assiette à bien et l&#39;aide de pinces appropriées fragments de taille sont choisis et placés dans d&#39;autres puits. 5 fragments de verre sont placés dans chaque puits de plaque de 6 puits. </li><li> Primaire des cellules de Schwann cultivées en flacon sont trypsinisées pendant 3 minutes en utilisant 0,1% de trypsine. Trypsinisées cellules de Schwann sont transférés à séparer le tube Falcon de 15 ml et centrifugées à 300 g pendant 5 minutes. Les cellules de Schwann sont remis en suspension à 2x 10 6 cellules / ml dans DMEM/10% SVF / 1% PSF. </li><li> 20 ul de gouttelettes cellules de Schwann suspension est ajoutée sur chaque lamelle 5 fragments placés dans 6 assiettes bien et les cellules sont incubées à 37 ° C CO, 7% 2 pendant 2 heures. </li><li> Après 2 heures, les cellules de Schwann sont attachés à la lamelle et fragments DMEM/10% SVF / 1% PSF (Complété avec 2 pm de la forskoline et de 10 pg / mL de BPE est ajouté à chaque puits afin que les fragments sont complètement couverts de moyenne) . </li><li> La plaque de 6 puits contenant les fragments sont ensuite placés dans l&#39;incubateur pendant 24-48 heures jusqu&#39;à ce que les fragments de verre sont complètement concouramment avec des cellules de Schwann. </li><li> Une fois les fragments sont confluentes avec des cellules de Schwann, chaque fragment est ramassé avec soin en utilisant une pince forte et trempée dans du HBSS pour enlever les cellules seules et ensuite le visage inversé vers le bas sur monocouche d&#39;astrocytes. </li><li> Chaque puits est ensuite recouvert avec 1 ml d&#39;DMEM/10% SVF / 1% de cellules de Schwann PSF et sont autorisés à migrer pendant 24 à 48 période. Durant cette période réactifs expérimentales telles que les facteurs de croissance peuvent être ajoutés afin d&#39;évaluer leur effet sur le nombre et la distance de migration des cellules de Schwann. </li><li> Après la période de migration, les fragments de verre sont fixés au fond de chaque puits en ajoutant 4% de paraformaldéhyde (PFA) pour 30 minutes. Après fixation, les cellules de Schwann immunocoloration pour la p75 (reconnaissant des récepteurs de faible affinité du NGF), puis les colorer à l&#39;aide ABC strepavidine kit-diaminobenzidine (DAB), pour permettre la visualisation des cellules de Schwann au microscope optique. </li></ol> 3. Les résultats représentatifs: Après 8-10 jours de Schwann-astrocytes co-cultures montrent une frontière nette entre les deux types cellulaires. Cette limite est plus prononcé si la forskoline et BPE est utilisé dans la culture comme la prolifération des cellules de Schwann est renforcée. Si aucune limite n&#39;est constatée pendant la période de 10 jours, le temps de la culture peut être encore augmenté jusqu&#39;à la frontières sont établies. Changer le rapport de la densité cellulaire est une autre façon d&#39;augmenter la formation de frontière. Un ratio de 3:1 de Schwann: la densité des cellules astrocytaires peuvent être utilisés lors de la préparation des suspensions cellulaires 8,18. Après immunomarquage pour identifier les cellules de Schwann et les astrocytes, des cellules d&#39;évaluer entremêlement une ligne peut être établi où la limite est établie et le nombre de cellules de Schwann migrant sur le territoire des astrocytes peuvent être comptés sous microscope à fluorescence. Voici deux exemples d&#39;images obtenues frontières, l&#39;un avec le bien-formé la ségrégation des cellules de Schwann et les astrocytes (figure 1A) et une où les cellules ne sont pas séparés et dans des territoires différents en raison de mauvaise technique (figure 1B). <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Figure 1" /> Test Boundary. Figure 1: Schwann-astrocytes interaction. A) Une journée 10 co-culture de dosage, en présence de forskoline et BPE à stimuler la prolifération de cellules de Schwann démontre une frontière nette entre les deux types de cellules (rouge = P75, vert = GFAP). B) Une journée 10 co-culture où la limite de dosage n&#39;est pas formé et les deux fronts de cellules mixtes. Cela est dû au mélange des deux gouttelettes tout en plaçant les gouttelettes de cellules de Schwann et des astrocytes côté de l&#39;autre lors de la préparation de la frontière (rouge = P75, vert = GFAP). <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Figure 2" /> Figure 2. Image représentative de monocouche d&#39;astrocytes confluents Migration des dosages d&#39;évaluer le mouvement d&#39;un seul type de cellules sur la surface d&#39;un autre type de cellule et donc d&#39;évaluer un phénomène différent de celui de la formation de frontière. La pratique est nécessaire pour assurer inversant les lamelles sur des monocouches ne conduit pas à des dommages à l&#39;cellule de Schwann couvert de fragments de verre et des monocouches d&#39;astrocytes par la pince forte. Afin d&#39;évaluer la migration des cellules de Schwann, la distance maximale de migration et le nombre de cellules qui migrent depuis le bord de la lamelle peut être mesurée au microscope optique. Ci-dessous est à l&#39;image de cellules de Schwann colorés avec des anticorps P75 utilisant une coloration DAB sur la surface des monocouches d&#39;astrocytes (figure 3). <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Figure 3" /> Test de migration figure 3. Utilisant test lamelle inversé. A) Les cellules de Schwann (brun, immunocolorées avec p75 anticorps) migrer loin du bord de la lamelle. La flèche indique la direction de la migration cellulaire loin de la lamelle.

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Les tests décrits ci-dessus ont été utilisés dans différentes études démontrant le rôle de multiples facteurs impliqués dans la formation frontière entre les astrocytes et Schwann limitées de migration de cellules de Schwann dans l&#39;environnement astrocytaire. Comprendre les mécanismes sous-jacents de ces événements est essentielle car elle permettrait le développement de stratégies visant à optimiser et améliorer l&#39;intégration des greffes de cellules de Schwann sui...

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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>
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analysis of schwann-astrocyte interactions using in vitro assays

Les cellules de Schwann sont l&#39;une des cellules couramment utilisés dans les stratégies de réparation suite à des blessures de la moelle épinière. Les cellules de Schwann sont capables de supporter la régénération axonale et la germination par les facteurs de croissance et en fournissant 1,2 sécrétant de promotion de croissance trois molécules d&#39;adhésion et des molécules de la matrice extracellulaire 4. En outre, ils myéliniser les axones démyélinisées au site de la lésion 5. Toutefois, suite à la transplantation, les cellules de Schwann ne migrent pas à partir du site de l&#39;implant et ne se mêlent avec les astrocytes hôte 6,7. Il en résulte la formation d&#39;une frontière nette entre les cellules de Schwann et les astrocytes, créant un obstacle pour les axones en croissance essayant de quitter l&#39;arrière du greffon dans le tissu hôte proximale et distale. Les astrocytes en contact avec les cellules de Schwann subissent également une hypertrophie et jusqu&#39;à réguler les molécules inhibitrices 8-13. Dans des essais in vitro ont été utilisées pour modéliser la cellule de Schwann-astrocytes interactions et ont été importantes dans la compréhension du mécanisme qui sous-tendent le comportement cellulaire. Ces tests in vitro comprennent test frontière, où une co-culture est faite en utilisant deux cellules différentes à chaque type de cellule d&#39;occupation des territoires différents avec seulement un petit écart qui sépare les deux fronts cellule. Comme les cellules se divisent et migrent, les deux fronts cellulaires se rapprocher les uns aux autres et finalement en collision. Cela permet au comportement des deux populations cellulaires à analyser à la frontière. Une autre variante de la même technique est de mélanger les deux populations cellulaires dans la culture et au fil du temps les deux types cellulaires séparer avec des cellules de Schwann groupées comme des îles dans entre les astrocytes ainsi que la création de plusieurs Schwann-astrocytes limites. Le second test utilisé dans l&#39;étude de l&#39;interaction des deux types cellulaires est le dosage de la migration cellulaires où le mouvement peut être suivi sur la surface de l&#39;autre type cellulaire monocouche 14,15. Ce dosage est communément appelé test lamelle inversé. Les cellules de Schwann sont cultivées sur des petits fragments de verre et ils sont inversés face cachée sur la surface de monocouches d&#39;astrocytes et de la migration est évaluée à partir du bord de la lamelle. Les deux dosages ont joué un rôle dans l&#39;étude des mécanismes sous-jacents impliqués dans l&#39;exclusion cellulaires et la formation de frontière. Certaines des molécules identifiées en utilisant ces techniques incluent le N-cadhérines 15, chondroïtine sulfate protéoglycanes (CSPG) 16,17, le FGF / Héparine 18, Eph / éphrines 19. Cet article vise à décrire de dosage et le dosage limite de migration en mode pas à pas et d&#39;élucider les éventuels problèmes techniques qui pourraient survenir.

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

Cet article vise à décrire de manière progressive l&#39;couramment utilisés<em&gt; In vitro</em&gt; Essais utilisés dans l&#39;étude de cellules de Schwann-asrtocyte interactions.

Analyse de Schwann-astrocytes interactions à l&#39;aide In Vitro Tests

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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Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.2K vues.  University of Cambridge. Cet article vise à décrire de manière progressive l'couramment utilisés In vitro Essais utilisés dans l'étude de cellules de Schwann-asrtocyte interactions.

Vidéo: 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.

Homogène de détection rapide des essais biologiques utilisant un support magnétique Système de modulation Biosensing

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

Recherche

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.

L&#39;analyse in vitro de PDZ-dépendants complexes macromoléculaires CFTR de signalisation

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

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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.

Dans la migration cellulaire in vitro et Invasion dosages

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' 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.

Analyse des réactions de transformation de l&#39;ARN utilisant des cellules des systèmes gratuits: 3 &#39;Fin de clivage de pré-ARNm substrats<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 agrégation essais utilisant la protéine tau hyperphosphorylée

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.

Analyse comparative de l&#39;hormone de croissance dans le sérum en utilisant SPRi, Nano-SPRi et dosages ELISA

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 ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Mesure du métabolisme énergétique chez Explanté tissu rétinien en utilisant l’analyse de Flux extracellulaire

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

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.

Effet des protéines fluorescentes sur des partenaires de Fusion utilisant les épreuves de toxicité Polyglutamine chez la levure

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.

Analyse du microbiote à l'aide de PCR en deux étapes et de la prochaine génération 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 ...