Ce travail a été soutenu par le Wellcome Trust nombre subvention [077108] attribué à GJW.

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L., Golan, D. E., Zhu, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+affinity+interaction+of+human+or+rat+T+cell+adhesion+molecule+CD2+with+its+ligand+aligns+adhering+membranes+to+achieve+high+physiological+affinity.">Low affinity interaction of human or rat T cell adhesion molecule CD2 with its ligand aligns adhering membranes to achieve high physiological affinity.</a> The Journal of biological chemistry. 272, (1997).</li><li>Wright, G. J., Martin, S., Bushell, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+identification+of+transient+extracellular+protein+interactions.">High-throughput identification of transient extracellular protein interactions.</a> Biochemical Society transactions. 38, (2010).</li><li>de Wildt, R. M., Tomlinson, I. M., Ong, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+receptor-ligand+pairs+by+capture+of+long-lived+multivalent+interaction+complexes.">Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, (2002).</li><li>Jiang, L., Barclay, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+leucocyte+surface+protein+interactions+by+high-throughput+screening+with+multivalent+reagents.">Identification of leucocyte surface protein interactions by high-throughput screening with multivalent reagents.</a> Immunology. 129, 55-55 (2010).</li><li>Urech, D. M., Lichtlen, P., Barberis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+growth+selection+system+to+detect+extracellular+and+transmembrane+protein+interactions.">Cell growth selection system to detect extracellular and transmembrane protein interactions.</a> Biochimica et biophysica acta. 1622, 117-117 (2003).</li><li>Voulgaraki, D., Mitnacht-Kraus, R., Letarte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivalent+recombinant+proteins+for+probing+functions+of+leucocyte+surface+proteins+such+as+the+CD200+receptor.">Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.</a> Immunology. 115, 337-337 (2005).</li><li>Wojtowicz, W. M., Wu, W., Andre, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vast+repertoire+of+Dscam+binding+specificities+arises+from+modular+interactions+of+variable+Ig+domains.">A vast repertoire of Dscam binding specificities arises from modular interactions of variable Ig domains.</a> Cell. 130, 1134-1134 (2007).</li><li>Martin, S., Sollner, C., Charoensawan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+Large+Extracellular+Protein+Interaction+Network+and+Its+Resolution+by+Spatiotemporal+Expression+Profiling.">Construction of a Large Extracellular Protein Interaction Network and Its Resolution by Spatiotemporal Expression Profiling.</a> Mol. Cell. Proteomics. 9, 2654-2654 (2010).</li><li>Crosnier, C., Bustamante, L. Y., Bartholdson, S. J., Bei, A. K., Theron, M., Uchikawa, M., Mboup, S., Ndir, O., Kwiatkowski, D. P., Duraisingh, M. T., Rayner, J. C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basigin+is+a+receptor+essential+for+erythrocyte+invasion+by+Plasmodium+falciparum.">Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum.</a> Nature. 480, 534-537 (2011).</li><li>Sollner, C., Wright, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+surface+interaction+network+of+neural+leucine-rich+repeat+receptors.">A cell surface interaction network of neural leucine-rich repeat receptors.</a> Genome biology. 10, R99-R99 (2009).</li></ol>

Les auteurs n&#39;ont rien à révéler.

Lorsque les cellules vivantes sont observées in vivo, leurs membranes plasmiques présentent des comportements très dynamiques: l&#39;extension et de rétraction des procédés membranaires comme ils entrent en contact et communiquer avec leurs 2 voisins. Les interactions physiques entre les protéines réceptrices membrane embarqués qui interviennent dans beaucoup de ces contacts ont évolué pour être extrêmement faible afin de permettre ce comportement très mobiles. Il a été estimé que les forces d&#39;interaction monomères aussi faibles que 50 uM pourrait être suffisante pour entraîner des interactions spontanées à des densités de récepteurs physiologiques 9 ce qui rend la détection de nouvelles interactions extrêmement difficiles 2,10. La méthode décrite ici AVEXIS fournit une méthode sensible pour détecter transitoire protéine extracellulaire: les interactions entre protéines qui peuvent être mises en œuvre d&#39;une manière évolutive avec un faible taux de faux positifs. Alors que d&#39;autres méthodes évolutives ont été développés pour détecter les interactions extracellulaires 11-15,un avantage de AVEXIS est que les paramètres expérimentaux tels que l&#39;appât et les activités de proies ont été quantifiés pour détecter même les plus faibles des interactions (t 1/2 ≤ 0,1 s) tout en conservant un faible taux de faux positifs 3. En outre, les procédures simplifiées et robuste de préparation des échantillons ont permis ce test à mettre en œuvre à une échelle beaucoup plus grande que d&#39;autres essais et nous avons récemment décrit un écran interaction systématique totalisant plus de ~ 16.500 interactions potentielles 16. Le dosage peut être effectué sur toute protéine pour laquelle il est possible d&#39;exprimer un fragment de protéine recombinante soluble. Cela comprend donc protéines qui contiennent un peptide signal N-terminale telle que le type I, GPI-liés et des protéines sécrétées. Nous avons récemment conçu une suite de vecteurs qui permettent aujourd&#39;hui d&#39;exprimer les protéines de type II comme appâts 17. Le test n&#39;est généralement pas adapté pour des protéines membranaires multipasses comme transporteur ioniques ou des pompes, car ils ont peu de chances d&#39;être correctement pliée en dehors du contexte d&#39;une membrane plasmique. Nous avons appliqué cette méthode à une variété de systèmes différents et ont exprimé avec succès des protéines extracellulaires de poisson-zèbre 3,16,18, humain, souris et P. falciparum pour les écrans d&#39;interaction. En ce qui concerne les ressources nécessaires pour mettre en place un écran AVEXIS, nous avons constaté que un goulot d&#39;étranglement important est la sélection, la construction, et le séquençage complet des plasmides d&#39;expression. Cet aspect de la méthode peut prendre jusqu&#39;à un an pour faire ~ 100 appâts et les constructions proies, mais, avec la baisse du coût de la synthèse de gènes, nous avons maintenant favoriser le commerce sous-traitance cet aspect du projet. Le système d&#39;expression mammifère avec un grand secouant incubateur de culture tissulaire (nous utilisons un INFORS HT Multitron Cell) permet à un seul chercheur à exprimer et à normaliser jusqu&#39;à ~ 100 protéines appâts et proies d&#39;un mois. Une fois que les protéines sont produites et normalisées, screening d&#39;interactions est rapide avec un maximum à douze plaques à 96 puits étant commodément traitées par jour. Le seul équipement sur mesure que nous avons mis au point pour faciliter la production d&#39;un tel grand nombre de protéines est un air comprimé à pistons entraînés qui est utilisé pour transmettre un maximum de cinq surnageants à travers un filtre 0.22μm en parallèle. La classe principale des interactions qui pourraient être manquées par rapport aux interactions attendues de la littérature (faux négatifs) sont des interactions homophiles, même si certaines de ces interactions peuvent être détectés 3. Dans les cas où les interactions attendues ne sont pas détectées, nous croyons que cela est dû à la formation des interactions homophiles par les protéines des proies (une proie &quot;masquage&quot; effet) ce qui l&#39;empêche de nouvelles interactions avec des protéines appâts immobilisés. La fréquence à laquelle les interactions sont détectées dépend du contenu de la bibliothèque protéine étant projeté. Comme un guide pour le lecteur, un écran de&gt; 30, 000 interactions au sein de la superfamille des immunoglobulines poisson zèbre abouti à 188 résultats positifs - une fréquence de 0,6% 3,16,18. Un chèque très rapide qui peut être effectuée pour vérifier les résultats positifs est de déterminer si l&#39;interaction peut être détecté dans les deux appât-proie orientations; qui est, peut être la même interaction détectée indépendamment du fait que les protéines de liaison sont présents sous forme soit d&#39;un appât ou une proie. Chaque fois que nous avons observé ce comportement alternatif, la liaison a ensuite été montré pour être précis, en démontrant la liaison directe saturable de protéines purifiées en utilisant résonance plasmonique de surface. Il convient de souligner que, parce que nous augmentons avidité de liaison par les protéines artificiellement pentamerising proies, nous ne considérons pas le dosage approprié pour comparer quantitativement les forces d&#39;interaction. Pour obtenir biophysiques paramètres de liaison d&#39;interactions que nous utilisons protéines monomériques et résonance plasmonique de surface. Enfin, une fois une interaction a été identifiée, le salutnature débit gh de l&#39;essai, il est relativement facile d&#39;adapter le dosage à l&#39;écran pour les réactifs tels que des anticorps ou des petites molécules qui bloquent l&#39;interaction.

<table border="1"><tbody><tr><td> Nom de réactif </td><td> Entreprise </td><td> Cat. Nombre </td><td> Commentaires </td></tr><tr><td> Freestyle médias </td><td> Invitrogen </td><td> 12338018 </td><td></td></tr><tr><td> Nitrocéfine </td><td> Calbiochem </td><td> 484400 </td><td></td></tr><tr><td> Des plaques de microtitrage SA-revêtues </td><td> Nunc </td><td> 734-1284 </td><td></td></tr><tr><td> OX68 anticorps </td><td> AbDSerotec </td><td> MCA1022R </td><td></td></tr><tr><td> Un tube de dialyse </td><td> Percer </td><td> PN68100 </td><td></td></tr><tr><td> Polymyxine B </td><td> Sigma </td><td> P4932 </td><td></td></tr><tr><td> D-biotine </td><td> Sigma </td><td> B4639-5G </td><td></td></tr><tr><td> Substrat-104 </td><td> Sigma </td><td> 1040-506 </td><td></td></tr><tr><td> Vivaspin-20 concentrateurs centrifuges </td><td> Sartorius </td><td> VS2002 </td><td></td></tr><tr><td> Des filtres de 0,22 um </td><td> Nalgene </td><td> 190-2520 </td><td></td></tr></tbody></table>

avidity-based extracellular interaction screening (avexis) for the scalable detection of low-affinity extracellular receptor-ligand interactions

Protéine extracellulaire: les interactions entre protéines sécrétées ou de la membrane entre-captifs protéines sont essentielles à la fois pour la cohésion initier la communication intercellulaire et d&#39;assurer au sein d&#39;organismes multicellulaires. Protéines prédit pour former des interactions extracellulaires sont codées par environ un quart des gènes de l&#39;homme 1, mais en dépit de leur importance et de l&#39;abondance, la majorité de ces protéines ont pas documentée partenaire de liaison. Principalement, cela est dû à leur indocilité biochimique: les protéines membranaires intégrées sont difficiles à solubiliser dans leur conformation native et contiennent structurellement importantes modifications post-traductionnelles. En outre, les affinités d&#39;interaction entre les protéines réceptrices sont souvent caractérisés par les forces d&#39;interaction extrêmement faibles (demi-vie &lt;1 seconde) excluant leur détection avec de nombreux couramment utilisés méthodes à haut débit 2. Ici, nous décrivons un essai, AVEXIS (avidité basée sur EXtracelluLAR Interaction écran) qui permet de surmonter ces défis techniques permettant la détection des interactions protéine-protéine très faibles (t 1/2 ≤ 0,1 sec) avec un faible taux de faux positifs 3. Le dosage est généralement mis en œuvre dans un format à haut débit afin de permettre le dépistage systématique de plusieurs milliers d&#39;interactions dans un format microplaque pratique (Fig. 1). Elle s&#39;appuie sur la production de protéines recombinantes solubles bibliothèques qui contiennent des fragments ectodomaine de récepteurs de surface cellulaire ou des protéines sécrétées dans lequel pour dépister les interactions et, par conséquent, cette approche est appropriée pour le type I, type II, liées au GPI récepteurs de surface cellulaire et sécrétées protéines, mais pas pour des protéines membranaires multipasses tels que les canaux ioniques ou des transporteurs. Les bibliothèques de protéines recombinantes sont produites en utilisant un système d&#39;expression pratique et de haut niveau chez les mammifères 4, pour s&#39;assurer que les modifications post-traductionnelles importantes telles que glycosylation et obligations disulfure sont ajoutés. Exprimées protéines recombinantes sont sécrétées dans le milieu et produit sous deux formes: un appât biotinylé qui peut être capturée sur une phase solide revêtue de streptavidine appropriée pour le dépistage, et une enzyme-étiqueté pentamerised (β-lactamase) proie. Le protéines appâts et proies sont présentés les uns aux autres dans un mode binaire pour détecter les interactions directes entre eux, semblables à un ELISA classique (Fig. 1). Le pentamerisation des protéines dans la proie est assurée par une séquence peptidique de la protéine de la matrice du cartilage oligomérique (COMP) et augmente la concentration locale des ectodomaines fournissant ainsi des gains significatifs avidité pour permettre des interactions, même très transitoires pour être détecté. En normalisant les activités de l &#39;appât et en proie à des niveaux prédéterminés avant le dépistage, nous avons montré que les interactions ayant monomères demi-vies de 0,1 sec peuvent être détectés avec de faibles taux de faux positifs 3.

Watch this Scientific Journal Video about Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions at JoVE.com

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS est un dosage à haut débit de protéines d&#39;interaction mis au point pour l&#39;écran systématiquement pour de nouveaux extracellulaires récepteur-ligand paires impliquées dans les processus de reconnaissance cellulaire. Il est spécialement conçu pour détecter les interactions entre protéines transitoires qui sont difficiles à identifier à l&#39;aide d&#39;autres approches à haut débit.

L&#39;avidité d&#39;un dépistage interaction extracellulaire (AVEXIS) pour la détection évolutive de faible affinité extracellulaires interactions récepteur-ligand

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

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Research

JoVE Journal

Biology

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

21.4K vues.  Wellcome Trust Sanger Institute. AVEXIS est un dosage à haut débit de protéines d'interaction mis au point pour l'écran systématiquement pour de nouveaux extracellulaires récepteur-ligand paires impliquées dans les processus de reconnaissance cellulaire. Il est spécialement conçu pour détecter les interactions entre protéines transitoires qui sont difficiles à identifier à l'aide d'autres approches à haut débit.

Vidéo: Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Profilage de masse Oligo (OLIMP) de polysaccharides extracellulaires

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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Biologie

Techniques for the Analysis of Extracellular Vesicles Using Flow Cytometry

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help regulate a diverse range of biological processes. Analysis of EVs using flow cytometry (FCM) has been notoriously difficult due to their small size and lack of discrete populations positive for markers of interest. Methods for EV analysis, while considerably improved over the last decade, are still a work in progress. Unfortunately, there is no one-size-fits-all protocol, and several aspects must be considered when determining the most appropriate method to use. Presented here are several different techniques for processing EVs and two protocols for analyzing EVs using either individual detection or a bead-based approach. The methods described here will assist with eliminating the antibody aggregates commonly found in commercial preparations, increasing signal&#8211;to-noise ratio, and setting gates in a rational fashion that minimizes detection of background fluorescence. The first protocol uses an individual detection method that is especially well suited for analyzing a high volume of clinical samples, while the second protocol uses a bead-based approach to capture and detect smaller EVs and exosomes.

Many different methods exist for the measurement of extracellular vesicles (EVs) using flow cytometry (FCM). Several aspects should be considered when determining the most appropriate method to use. Two protocols for measuring EVs are presented, using either individual detection or a bead-based approach.

Techniques pour l&#39;analyse des vésicules extracellulaire par cytométrie en flux

Extracellular Vesicles (EVs) are small, membrane-derived vesicles found in bodily fluids that are highly involved in cell-cell communication and help ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

Mesure de extracellulaire Ion flux Utilisation de l&#39;Ion sélectif microélectrodes Technique d&#39;auto-référencement

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Une expression efficace de cellules de mammifères et en une seule étape de purification extracellulaire glycoprotéines à la cristallisation

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Microscopie électronique à balayage du tissu macéré pour visualiser la matrice extracellulaire

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

Un rapide, méthode évolutive pour l&#39;isolement, l&#39;étude fonctionnelle, et l&#39;analyse de la matrice extracellulaire de cellules dérivées

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Mise en place d’un pH extracellulaire acide système de Culture

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

A BW Reporter System for Studying Receptor-Ligand Interactions

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native receptor and the ligand are challenging since the ligand of a specific receptor may be unknown and experimental procedures with the native ligand can be technically complicated. To address these obstacles, we describe a reporter system to detect the binding and activation of a specific receptor by a ligand of interest. In this reporter system, the extracellular domain of a specific receptor is conjugated to mouse CD3&#950; and this chimeric protein is then expressed in mouse BW cells. These transfected BW cells can then be incubated with different targets (e.g., cells or antibodies). Activation of a transfected receptor leads to the secretion of mouse interleukin-2 (mIL-2) which can be detected by enzyme-linked immunosorbent assay (ELISA). This reporter system has the advantages of being sensitive and specific to a single receptor. In addition, the activation level of a specific receptor can easily be quantified and can be used even in cases where the ligand of the receptor is unknown. This system has been implemented successfully in many of our studies to characterize receptor-ligand interactions. We have recently employed this system to study the activation of human Fc&#947; receptors (Fc&#947;Rs) by different monoclonal anti-CD20 antibodies in clinical use.

This protocol describes how to establish a reporter system that can be used to identify and quantify receptor-ligand interactions.

Un système de BW rapporteur pour étudier les Interactions Ligand-récepteur

Interactions between receptors and ligands constitute a fundamental biological process. However, direct experiments with cells that express the native ...

Nanoparticle Tracking Analysis for the Quantification and Size Determination of Extracellular Vesicles

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. There are many different methods for EV isolation, each with distinct advantages and disadvantages that affect the downstream yield and purity of EVs. Thus, characterizing the EV prep isolated from a given source by a chosen method is important for interpretation of downstream results and comparison of results across laboratories. Various methods exist for determining the size and quantity of EVs, which can be altered by disease states or in response to external conditions. Nanoparticle tracking analysis (NTA) is one of the prominent technologies used for high-throughput analysis of individual EVs. Here, we present a detailed protocol for quantification and size determination of EVs isolated from mouse perigonadal adipose tissue and human plasma using a breakthrough technology for NTA representing major advances in the field. The results demonstrate that this method can deliver reproducible and valid total particle concentration and size distribution data for EVs isolated from different sources using different methods, as confirmed by transmission electron microscopy. The adaptation of this instrument for NTA will address the need for standardization in NTA methods to increase rigor and reproducibility in EV research.

We demonstrate how to use a novel nanoparticle tracking analysis instrument to estimate the size distribution and total particle concentration of extracellular vesicles isolated from mouse perigonadal adipose tissue and human plasma.

Analyse de suivi des nanoparticules pour la quantification et la détermination de la taille des vésicules extracellulaires

The physiological and pathophysiological roles of extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving ...

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

Test d’absorption des vésicules extracellulaires par analyse d’imagerie au microscope confocal

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...