Nous remercions M. Wolf Frommer (Carnegie Institution for Science, l&#39;Université de Stanford) d&#39;avoir bien voulu nous fournir le glucose FRET plasmide et le Dr Uwe Ludewig (Université Hohenheim) pour fournir de bien vouloir la glutamine FRET construction dans l&#39;usine vecteur d&#39;expression pUTKan. AB est financé par le programme des Bourses d&#39;études prioritaires BBSRC ciblée. La CK et KP sont pris en charge par des bourses RCUK en traitement Biopharmaceuticals. CK tient également à remercier Lonza Biologics pour leur soutien financier. Le Centre pour la biologie synthétique et d&#39;innovation est généreusement soutenu par l&#39;EPSRC.

Une. Cell Line Revival et entretien <ol><li> Ressusciter des cellules CHO dans 9 ml de milieu CD-CHO supplémenté avec 8 mM de L-glutamine et de 10 ml / L supplément 100x hypoxanthine / thymidine (milieu de croissance complet). </li><li> Centrifuger à 100 g pendant 5 min. </li><li> Remettre en suspension les cellules dans 10 ml de milieu de croissance frais complet. </li><li> Retirer un échantillon de 1 ml et déterminer la concentration de cellules viables par microscopie optique en utilisant le procédé d&#39;exclusion de colorant bleu trypan dans un hémocytomètre. </li><li> Initier une culture à une densité de semis de 3 x 10 5 cellules / ml dans 125 ml secouer les flacons. </li><li> Maintenir la culture dans un incubateur à 37 ° C, une atmosphère humidifiée de 5% de CO 2, sur une table d&#39;agitation orbital tournant à 120 tours par minute. </li><li> Repiquer tous les 3-4 jours dans le milieu de croissance complet à une densité de semis de 2 x 10 5 cellules / ml. </li></ol> 2. La transfection de la lignée cellulaire avec le plasmide Contenant le biocapteur Gene <ol><li> Préparer l&#39;ADN de plasmide en utilisant un kit de purification de plasmide à grande échelle. </li><li> Maintenir les cellules dans des conditions appropriées (37 ° C, 5% CO 2) de 12 à 24 h avant la transfection afin d&#39;assurer les cellules se divisent activement au moment de la transfection. </li><li> Compter les cellules à l&#39;aide de la méthode d&#39;exclusion du colorant bleu trypan, puis préparer un volume de travail de 20 ml de culture cellulaire dans un flacon à secousses de 125 ml à une concentration de 1 x 10 6 cellules / ml. </li><li> Utilisation d&#39;un kit de transfection approprié pour la lignée cellulaire utilisée. L&#39;ADN plasmidique à ratio des réactifs de transfection sera fonction de la lignée cellulaire et le vecteur. Il est recommandé que cette est optimisée en testant une gamme de rapports au sein de la gamme spécifiées par le fabricant avant de procéder à des transfections finales. Maintenir en outre au moins une culture témoin négatif contenant les cellules, mais pas d&#39;ADN. </li><li> Incuber pendant 4 jours en mode statique, puis les transférer à un Shakiplate-forme ng tournant à 125 rpm. </li><li> Ajouter l&#39;antibiotique approprié pour la sélection de plasmide à une concentration appropriée telle que déterminée par une courbe de destruction. Dans cette étude, la zéocine a été ajouté à une concentration finale de 400 pg / ml pour la sélection des cellules transfectées. </li><li> Changer de support à un intervalle de temps approprié pour la lignée cellulaire, ajoutant antibiotique à chaque fois, jusqu&#39;à ce que les cellules de contrôle et sont morts. </li><li> Utilisez les puits les plus confluentes de progresser vers les cultures agitées. </li><li> Établir une banque de cellules par congélation des cellules dans des flacons cryogéniques contenant 10 7 cellules viables dans 1 ml de mélange gel. Dans ce travail, il s&#39;agit d&#39;un milieu de croissance de 92,5% et 7,5% de diméthylsulfoxyde. </li></ol> 3. Batch et Fed-batch croissance cellulaire Curve <ol><li> Suivez les instructions pour l&#39;entretien des cellules ci-dessus pour établir des cultures de cellules triple des cellules CHO transfectées dans des flacons de 250 ml secouer avec un volume de travail de 50 ml. </li><li> Maintenir la cultures dans un incubateur humidifié de cellule à 37 ° C, avec 5% de CO 2, sur une plate-forme orbitale secouant tournant à 125 tours par minute, et retirer 4,1 ml des échantillons de chaque culture de plus en plus à intervalles de 24 heures. </li><li> Utiliser 100 pl de l&#39;échantillon pour déterminer la concentration cellulaire et la viabilité des cellules viables avec la méthode d&#39;exclusion au bleu de trypan colorant. </li><li> Répéter jusqu&#39;à ce que la concentration de cellules viables est réduit à zéro. </li><li> Répéter pour les cultures de cellules fed-batch en complétant avec la quantité appropriée de glucose ou de la glutamine au jour 6 de restaurer leurs concentrations à leurs valeurs initiales de 36 mm et 4 mm, respectivement. Il est recommandé que les cultures de contrôle supplémentaires sont également maintenues, qui doivent être alimentés avec le même volume (12 ml dans le cas présent) de l&#39;eau pure. </li></ol> 4. FRET Ratio mesures <ol><li> Prendre 2 ml des échantillons quotidiens retirées à l&#39;étape 3.2 ci-dessus et centrifuger à 100 g pendant 5 min à 4 ° C. </li><li> Resuspterminé le culot de cellules dans 2 ml de glace froide saline tamponnée au phosphate (PBS). Transférer cette dans une plaque à 6 puits et ajouter un échantillon témoin ne contenant pas de cellules pour un bien. </li><li> Mesurer les niveaux de fluorescence bleue et jaune immédiatement à une longueur d&#39;onde d&#39;excitation de 430/435 nm et d&#39;émission des longueurs d&#39;onde de 465/435 nm (bleu) et 520/510 nm (jaune). </li><li> Calculer les rapports de FRET (ratio de fluorescence jaune détecté sur fluorescence bleue détecté). </li></ol> 5. Métabolite dosages <ol><li> Prendre 2 ml des échantillons quotidiens retirées à l&#39;étape 3.2 ci-dessus et centrifuger à 100 g pendant 5 min à 4 ° C. Retirer le surnageant. </li><li> Remettre en suspension le culot cellulaire dans 3 ml de PBS glacé et centrifuger à nouveau à 100 x g pendant 5 min. </li><li> Eliminer le surnageant et remettre en suspension le culot cellulaire dans 3 ml de PBS refroidi à la glace comme ci-dessus. Soniquer l&#39;échantillon 5x pendant 3 min chacune à une impulsion de 15 secondes sur 15 secondes et éteint. À ce stade, la cellule supplémentairects peuvent être congelées si vous le souhaitez. </li><li> Utilisez un kit de dosage du glucose approprié selon les instructions du fabricant sur des échantillons d&#39;extraits cellulaires pour déterminer la concentration intracellulaire de glucose (voir tableau des matériaux). Remarque: Utilisez des normes appropriées pour construire une courbe standard. Dans ce travail, la concentration de normes variait entre 0 à 100 mM. </li><li> À partir des valeurs pour les normes, construire la courbe d&#39;étalonnage et de trouver l&#39;équation linéaire de meilleur ajustement (dans ce cas: y = 1310.51x - 723,43 où y est la lecture de l&#39;absorbance et x est la concentration de glucose). </li><li> Calculer les concentrations de glucose en utilisant la courbe d&#39;étalonnage et en tenant compte de la dilution. La concentration intracellulaire de glucose peut alors être calculée en utilisant l&#39;équation suivante: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> Pour les cellules CHO, le volume de la cellule unique a été calculée en utilisant un diamètre de cellule de 12 uM 12 </sup&gt;. </li><li> Utilisez un kit de dosage de la glutamine appropriée selon les instructions du fabricant sur des échantillons d&#39;extraits cellulaires pour déterminer la concentration de glutamine intracellulaire (voir tableau des matériaux). Remarque: Utilisez des normes appropriées pour construire une courbe standard. Dans ce travail, la concentration des normes variait entre 0-2 mM. </li><li> Comme dans 5.5 ci-dessus, la construction de la courbe d&#39;étalonnage (dans ce cas y = + 17,1 203.1x où y est la mesure d&#39;absorbance et x est la concentration de glucose) </li><li> En utilisant la courbe d&#39;étalonnage, calculer la concentration de glutamine des échantillons, en tenant compte de la dilution. La concentration de glutamine intracellulaire peut être calculée en utilisant l&#39;équation 1 ci-dessus. </li></ol>

<ol>
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Les auteurs n&#39;ont rien à révéler.

Les FIBS permettent in vivo et la quantification in situ des molécules clés dans, dans ce cas des nutriments limitant la croissance, éliminer les incertitudes découlant de la trempe et de l&#39;extraction des méthodes. Les résultats suggèrent qu&#39;il existe une bonne corrélation entre le signal de FIBS et les concentrations intracellulaires de l&#39;ordre de 1-5 mM pour le glucose et 0,3-2 mM pour la glutamine. Dans le lot CHO culture cellulaire, ces concentrations sont rencontrées ...

suivi de métabolite a diverses applications dans les procédés biotechnologiques, y compris le processus de développement, les médias et la conception d&#39;alimentation, et de l&#39;ingénierie métabolique. Diverses méthodes sont disponibles pour la mesure de concentration à l&#39;aide d&#39;une enzymatique, chimique 2, ou des dosages de liaison 3. Une solution intéressante est l&#39;utilisation de fluorescence, dans des capteurs biologiques in vivo que les reporters de la concentration intracellulaire des métabolites principaux à base de protéines. Dans les essais de volume ultra-bas, la fluorescence est un outil pratique que la miniaturisation améliore réellement le taux de 4,5 signal-sur-bruit et des capteurs à base de protéines peut être génétiquement codé sens qu&#39;aucun réactifs exogènes sont nécessaires pour l&#39;analyse des métabolites. Förster Resonance Energy Transfer (FRET) biocapteurs sont constitués de deux protéines fluorescentes reliés par un domaine de liaison du ligand. FRET est un transfert non radiatif de l&#39;énergie à partir d&#39;un donneur de photo-excitation à un accepteur fluorescent molécule locat<html> ed à proximité (&lt;100). Le clivage de la liaison du ligand ou provoque un changement conformationnel dans le capteur, ce qui, à son tour, induit un changement dans la proximité des fluorophores, ce qui conduit à un changement de l&#39;efficacité de FRET mesuré par le changement dans le spectre d&#39;émission. Des capteurs biologiques intégrés (FRET) sont constitutivement FIBS produits dans la lignée de cellules et leurs caractéristiques spectrales changent en fonction de la concentration du métabolite dans la cellule. FIBS ont des temps de réponse rapide qui les rend idéales pour prendre des mesures pour les modèles dynamiques 6. Applications précédentes comprennent la surveillance simples 7-9 et plusieurs métabolites 10 et fournir des données sur la répartition spatio-temporelle 11. FIBS peuvent être créés dans deux configurations: Apo-Max, où la liaison du ligand perturbe la proximité des fluorophores abaissement transfert d&#39;énergie et Apo-Min, où la liaison du ligand apporte les deux fluorophores en contact plus étroit (figure 1). _content &quot;&gt; Dans ce travail, un protocole est présenté pour la construction d&#39;ovaire de hamster chinois (CHO), des lignées cellulaires qui expriment de manière constitutive un FIBS pour un métabolite, le glucose ou la glutamine. Une méthodologie est établie pour l&#39;étalonnage du capteur in vivo pour permettre à l&#39;avenir quantitative des mesures de concentration de métabolite intracellulaire, tel que présenté dans la figure 2. Sur cette base, la concentration intracellulaire de glucose ou de la glutamine peuvent être déterminées en fed-batch CHO cultures de cellules, dans laquelle les deux éléments nutritifs ont été ajoutés à des concentrations élevées en cours de fabrication. Les résultats démontrent que l&#39;utilisation du signal de fluorescence à partir des FIBS et la courbe d&#39;étalonnage construite précédemment, avec précision la prédiction de la concentration intracellulaire est possible, comme l&#39;a confirmé par un test enzymatique indépendante. Cette méthode offre des avantages substantiels par rapport aux technologies d&#39;analyse de courant, car il est peu coûteux non invasive et rapide, donnant un signal en temps réel des FIBS qui peuvent être monitored tout au long de la culture.

<table border="0" cellpadding="0" cellspacing="0" width="936">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
</table>

fibs-enabled noninvasive metabolic profiling

À l&#39;ère de la biologie computationnelle, de nouveaux systèmes expérimentaux à haut débit sont nécessaires afin de remplir et d&#39;affiner les modèles de sorte qu&#39;ils peuvent être validées à des fins prédictives. Idéalement, de tels systèmes seraient faible volume, ce qui exclut d&#39;échantillonnage et les analyses destructives lorsque des données de cours du temps doivent être obtenus. Ce qui est nécessaire est un outil de surveillance in situ qui peuvent rapporter les informations nécessaires en temps réel et de façon non invasive. Une solution intéressante est l&#39;utilisation de fluorescence, dans des capteurs biologiques in vivo que les reporters de concentrations intracellulaires à base de protéines. Une classe particulière de biocapteurs in vivo qui ont trouvé des applications dans métabolite quantification est basée sur le transfert d&#39;énergie par résonance Förster (FRET) entre deux protéines fluorescentes reliés par un domaine de liaison du ligand. FRET capteurs biologiques intégrés (mensonges) sont constitutivement produites dans la lignée cellulaire, ils ont un temps de réponse rapide et leur cha spectraletiques changer en fonction de la concentration de métabolite dans la cellule. Dans le présent document, le procédé de construction d&#39;ovaire de hamster chinois (CHO), des lignées cellulaires qui expriment de manière constitutive un FIBS de glucose et de la glutamine et l&#39;étalonnage des FIBS in vivo dans une culture cellulaire par lots afin de permettre la quantification future de la concentration en métabolite intracellulaire est décrite. Les données de fed-batch cultures de cellules CHO démontre que les FIBS a pu dans chaque cas, de détecter la variation résultante de la concentration intracellulaire. En utilisant le signal fluorescent à partir des FIBS et la courbe d&#39;étalonnage construite précédemment, la concentration intracellulaire a été déterminée avec précision telle que confirmée par un essai enzymatique indépendante.

Un aperçu de la méthodologie est présentée dans la figure 2. Dans le travail présenté ici, les cellules CHO ont été transfectées avec le vecteur d&#39;FIBS et des lignées cellulaires stables ont été sélectionnées à un antibiotique pression de 400 pg / ml de zéocine. Deux lignées cellulaires stables exprimant de manière constitutive séparées de glucose et de glutamine capteurs ont donc été créés. Figure 1 représente les configurations des deux biocapteurs utilis?...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

Une description de la façon d&#39;étalonner les capteurs de transfert Förster Resonance Energy intégrés biologiques (mensonges) pour le profilage métabolique in situ est présentée. Les FIBS peuvent être utilisés pour mesurer les niveaux de métabolites intracellulaires aider de manière non invasive dans le développement de modèles métaboliques et criblage à haut débit de conditions de bioprocédés.

FIBS-permis non invasive au profil métabolique

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K vues.  Imperial College London.  Une description de la façon d'étalonner les capteurs de transfert Förster Resonance Energy intégrés biologiques (mensonges) pour le profilage métabolique in situ est présentée. Les FIBS peuvent être utilisés pour mesurer les niveaux de métabolites intracellulaires aider de manière non invasive dans le développement de modèles métaboliques et criblage à haut débit de conditions de bioprocédés. 

Vidéo: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

Haute profilage de microARN Débit: Multiplex Optimisé qRT-PCR à l&#39;échelle nanolitres sur l&#39;IFC de Fluidigm ArrayTM dynamique

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Recherche

Bio-ingénierie

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

In situ à la compression de chargement et d&#39;imagerie non invasive corrélative de l&#39;os-ligament parodontal-dent Fort commune

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Quantification et Taille-profilage des vésicules extracellulaires Utilisation accordable résistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Mécano-profilage facile et précis sur Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Glycosylation métabolique de l’acide sialique à l’aide de N-acyl-modification Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

Soutien métabolique des excisées, les tissus de cerveau vivants lors de l’Acquisition de la microscopie par résonance magnétique

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry : Une approche puissante et non invasive pour étudier le métabolisme énergétique cellulaire

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nano-capteurs pour détecter l’activité protéase In Vivo pour le diagnostic non invasif

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Mesure des métalloprotéinases de la matrice cellulaire Global et l’activité métabolique des Hydrogels 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Un modèle de culture matrix-adipocyte extracellulaire humaine 3D pour étudier matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...