Ringraziamo il professor Lupo Frommer (Carnegie Institution for Science, Stanford University) per averci gentilmente fornito con il glucosio FRET plasmide e il Dr. Uwe Ludewig (Hohenheim University) per fornire gentilmente la glutammina FRET costrutto nel pUTKan pianta vettore di espressione. AB è finanziato dal programma di Borse di studio prioritari BBSRC mirata. Sia CK e KP sono supportati da RCUK Borse in Biopharmaceuticals Processing. CK desidera inoltre ringraziare Lonza Biologics per il loro sostegno finanziario. Il Centro per la biologia sintetica e l&#39;innovazione è generosamente supportato dal EPSRC.

1. Cella linea Revival e manutenzione <ol><li> Revive cellule CHO in 9 ml CD-CHO medio integrato con 8 mM L-glutammina e 10 ml / L integratore 100x ipoxantina / timidina (terreno di coltura completo). </li><li> Centrifugare a 100 xg per 5 min. </li><li> Risospendere le cellule in 10 ml di fresco mezzo di crescita integrale. </li><li> Rimuovere un campione di 1 ml e determinare la concentrazione di cellule vitali mediante microscopio ottico utilizzando il metodo esclusione del colorante blu tripano in un emocitometro. </li><li> Avviare una coltura ad una densità di semina di 3 x 10 5 cellule / ml in 125 ml agitare palloni. </li><li> Mantenere la cultura in un incubatore a 37 ° C, atmosfera umidificata al 5% CO 2, su un agitatore orbitale rotante a 120 rpm. </li><li> Subcuture ogni 3-4 giorni nel mezzo di crescita completo ad una densità di semina di 2 x 10 5 cellule / ml. </li></ol> 2. La trasfezione della linea cellulare con il plasmide Containing il biosensore Gene <ol><li> Preparare DNA plasmidico utilizzando un kit di purificazione plasmide larga scala. </li><li> Mantenere celle in condizioni appropriate (37 ° C, 5% CO 2) 12-24 ore prima della trasfezione per garantire le cellule sono attivamente dividono al momento della transfezione. </li><li> Contare le cellule utilizzando il metodo di esclusione del colorante blu tripano, quindi preparare un volume utile di 20 ml di coltura cellulare in un matraccio da 125 ml agitare ad una concentrazione di 1 x 10 6 cellule / ml. </li><li> Utilizzare un kit di transfezione adatto per la linea cellulare in uso. Il DNA plasmidico rapporto reagenti di transfezione sarà dipendente dalla linea cellulare e il vettore. Si raccomanda che questa è ottimizzato testando una serie di rapporti entro l&#39;intervallo specificato dal fabbricante prima di effettuare le trasfezioni finali. Anche mantenere almeno una coltura di controllo negativo contenente le cellule ma non DNA. </li><li> Incubare per 4 giorni in modalità statica e quindi il trasferimento a un Shakipiattaforma ng rotante a 125 rpm. </li><li> Aggiungere l&#39;antibiotico appropriato per la selezione plasmide ad una concentrazione adatta come determinato da una curva uccisione. In questo studio, Zeocin stato aggiunto ad una concentrazione finale di 400 mg / ml per la selezione delle cellule trasfettate. </li><li> Cambiare media a un intervallo di tempo adeguato per la linea cellulare, aggiungendo antibiotici ogni volta, fino a quando le cellule in pozzetto di controllo sono morti. </li><li> Utilizzare i pozzi più confluenti a progredire a scuotere le culture. </li><li> Istituire una banca di cellule congelando le cellule in fiale criogeniche contenenti 10 7 cellule vitali in 1 ml di mix di congelamento. In questo lavoro, questo è costituito da 92,5% mezzo di crescita e 7,5% dimetilsolfossido. </li></ol> 3. Batch e fed-batch curva di crescita cellulare <ol><li> Seguire le istruzioni per la manutenzione cella sopra stabilire colture cellulari triplicato di cellule CHO trasfettate in 250 ml agitare beute con un volume utile di 50 ml. </li><li> Mantenere la culres in un incubatore umidificato cella a 37 ° C, con 5% di CO 2, su un agitatore orbitale rotante a 125 rpm, e rimuovere 4,1 ml campioni di ciascuna coltura in crescita ad intervalli di 24 hr. </li><li> Utilizzare 100 ml di campione per determinare la fattibilità di concentrazione cellula e cellula vitale con l&#39;azzurro metodo di esclusione Tripan. </li><li> Ripetere finché la concentrazione di cellule vitali è ridotto a zero. </li><li> Ripetere l&#39;operazione per le colture cellulari fed-batch che completa con la giusta quantità di glucosio o di glutammina il giorno 6 a ripristinare le concentrazioni ai valori iniziali di 36 e 4 mm, rispettivamente. Si raccomanda che colture di controllo aggiuntive sono anche mantenuti, che devono essere alimentati con lo stesso volume (12 ml in questo caso) di acqua pura. </li></ol> 4. FRET Rapporto Misure <ol><li> Prendere 2 ml dei campioni giornalieri rimosse al punto 3.2 sopra e centrifugare a 100 xg per 5 minuti a 4 ° C. </li><li> Resuspterminato il pellet di cellule in 2 ml di ghiaccio freddo fosfato salino (PBS). Trasferire questa in una piastra da 6 pozzetti e aggiungere il campione in bianco che non contiene cellule ad un pozzetto. </li><li> Misurare i livelli di fluorescenza blu e giallo subito a una lunghezza d&#39;onda di eccitazione di 430/435 nm e di emissione lunghezze d&#39;onda di 465/435 nm (blu) e 520/510 nm (giallo). </li><li> Calcolare i rapporti FRET (rapporto di fluorescenza gialla rilevato sopra fluorescenza blu rilevato). </li></ol> 5. Metabolita Saggi <ol><li> Prendere 2 ml dei campioni giornalieri rimosse al punto 3.2 sopra e centrifugare a 100 xg per 5 minuti a 4 ° C. Rimuovere il surnatante. </li><li> Risospendere il pellet di cellule in 3 ml di PBS freddo ghiaccio e centrifugare di nuovo a 100 xg per 5 min. </li><li> Rimuovere il surnatante e risospendere il pellet cellulare in 3 ml di PBS freddo ghiaccio come sopra. Sonicare il 5x campione per 3 minuti ciascuno in un impulso di 15 secondi su e 15 sec off. A questo punto, la cella supplementarects possono essere congelati se lo si desidera. </li><li> Utilizzare un kit di analisi glucosio appropriato secondo le istruzioni del fabbricante su campioni di estratti cellulari per determinare la concentrazione di glucosio intracellulare (vedi tabella dei materiali). Nota: utilizzare norme adeguate per costruire una curva standard. In questo lavoro, la concentrazione degli standard varia tra 0-100 mm. </li><li> Usando le letture per gli standard, costruire la curva standard e trovare l&#39;equazione lineare di best fit (in questo caso: y = 1310.51x - 723,43 dove y è la lettura di assorbanza e x è la concentrazione di glucosio). </li><li> Calcolare le concentrazioni di glucosio utilizzando la curva standard e prendendo la diluizione in considerazione. La concentrazione di glucosio intracellulare può quindi essere calcolato usando questa equazione: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> Per le cellule CHO, il volume singola cella è stato calcolato utilizzando una cella diametro di 12 pM 12 </sup&gt;. </li><li> Utilizzare un kit di analisi glutammina appropriato secondo le istruzioni del fabbricante su campioni di estratti cellulari per determinare la concentrazione intracellulare glutammina (vedi tabella dei materiali). Nota: utilizzare norme adeguate per costruire una curva standard. In questo lavoro, la concentrazione degli standard variava tra 0,2 mm. </li><li> Come in 5.5 sopra, la curva di calibrazione (in questo caso y = 203.1x + 17.1 dove y è la lettura dell&#39;assorbanza e x è la concentrazione di glucosio) </li><li> Usando la curva standard, calcolare la concentrazione glutammina dei campioni, tenendo conto della diluizione. La concentrazione intracellulare glutamina può essere calcolato utilizzando l&#39;Equazione 1 di cui sopra. </li></ol>

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Le FIBS permettono in vivo e in situ quantificazione di molecole chiave, in questo caso nutrienti crescita limitativo, eliminando incertezze derivanti da tempra e metodi di estrazione. I risultati suggeriscono che vi è una buona correlazione tra il segnale FIBS e le concentrazioni intracellulari nell&#39;intervallo 1-5 mM di glucosio e 0,3-2 mM di glutammina. Nel lotto coltura cellulare CHO, queste concentrazioni si incontrano nelle fasi di declino esponenziale, stazionarie, e le prime. Le fasi espone...

Monitoraggio metabolita ha varie applicazioni in bioprocessi, compreso lo sviluppo di processo, media e il design dei mangimi, e l&#39;ingegneria metabolica. Vari metodi sono disponibili per le misure di concentrazione attraverso l&#39;uso di 1 enzimatica, chimico 2, o saggi di legame 3. Un&#39;opzione interessante è l&#39;uso di fluorescente, sensori biologici in vivo come reporter della concentrazione intracellulare di metaboliti principali a base di proteine. In saggi ultra-basso volume, fluorescenza è uno strumento utile come miniaturizzazione migliora anche il rapporto 4,5 segnale-rumore e sensori a base di proteine ​​può essere geneticamente codificato senso che non reagenti esogeni sono necessari per l&#39;analisi metabolita. Trasferimento Förster Resonance Energy (FRET) biosensori costituiti da due proteine ​​fluorescenti collegati da un dominio di legame al ligando. FRET è un trasferimento nonradiative di energia da un donatore foto-eccitati per un accettore fluorescente molecola Locat<html> Ed in prossimità (&lt;100). Ligando clivaggio o vincolante provoca un cambiamento conformazionale nel sensore, che, a sua volta, induce un cambiamento in prossimità dei fluorofori, portando ad un cambiamento di efficienza FRET misurata dalla variazione dello spettro di emissione. Sensori biologici FRET integrati (FIBS) sono costitutivamente prodotte all&#39;interno della linea cellulare e le loro caratteristiche spettrali variare in funzione della concentrazione del metabolita all&#39;interno della cellula. FIBS hanno tempi di risposta veloci che li rende ideali per prendere le misure per i modelli dinamici 6. Applicazioni precedenti includono il monitoraggio singole 7-9 e più di 10 metaboliti e fornendo dati sulla distribuzione spazio-temporale 11. FIBS possono essere creati in due configurazioni: Apo-Max, dove legame del ligando interrompe la vicinanza dei fluorofori abbassamento trasferimento di energia e Apo-Min, dove legame ligando porta i due fluorofori in più stretto contatto (Figura 1). _content &quot;&gt; In questo lavoro, un protocollo è presentato per la costruzione di ovaio di criceto cinese (CHO) linee cellulari che esprimono costitutivamente un FIBS per un metabolita, glucosio o glutammina. Una metodologia è stabilito per la calibrazione del sensore in vivo per abilitare futuro quantitativa misure di concentrazione metabolita intracellulare, come illustrato nella figura 2. Sulla base di questo, la concentrazione intracellulare di glucosio o glutammina può essere determinato in fed-batch colture di cellule CHO, a cui i due nutrienti sono stati aggiunti in alte concentrazioni in-process. I risultati dimostrano che utilizzando il segnale fluorescente dalle FIBS e la curva di taratura precedentemente costruita, accuratamente previsione della concentrazione intracellulare è possibile, come confermato mediante saggio enzimatico indipendente. Questo metodo offre notevoli vantaggi rispetto alle attuali tecnologie analitiche perché è invasiva, a basso costo e veloce, dando un segnale in tempo reale dei FIBS che possono essere monitored tutta la cultura.

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	<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

Nell&#39;era della biologia computazionale, i nuovi sistemi sperimentali ad alto rendimento sono necessarie per compilare e perfezionare i modelli in modo che possano essere convalidati a fini predittivi. Idealmente tali sistemi sarebbero basso volume, che preclude campionamento e analisi distruttive quando i dati del corso tempo si vogliono ottenere. Ciò che occorre è un strumento di monitoraggio in situ che possono comunicare le informazioni necessarie in tempo reale e in maniera non invasiva. Un&#39;opzione interessante è l&#39;uso di fluorescente, sensori biologici in vivo segnalanti concentrazioni intracellulari base di proteine. Una classe particolare di biosensori in vivo che ha trovato applicazioni in metabolita quantificazione si riferiscono al trasferimento Förster Resonance Energy (FRET) tra due proteine ​​fluorescenti collegati da un dominio di legame al ligando. FRET sensori biologici integrati (FIBS) sono costitutivamente prodotte all&#39;interno della linea cellulare, hanno tempi di risposta veloci e il loro cha spettralestiche variare in funzione della concentrazione del metabolita all&#39;interno della cellula. In questo documento, il metodo per la costruzione di ovaio di criceto cinese (CHO) linee cellulari che esprimono costitutivamente un FIBS per il glucosio e glutammina e calibrare le FIBS in vivo in coltura cellulare in batch per consentire futuro quantificazione della concentrazione di metabolita intracellulare è descritto. I dati di fed-batch colture di cellule CHO dimostra che i FIBS è stato in grado in ogni caso a rilevare il conseguente cambiamento della concentrazione intracellulare. Utilizzando il segnale fluorescente dalle FIBS e la curva di taratura precedentemente costruita, la concentrazione intracellulare è stata determinata con precisione come confermato mediante saggio enzimatico indipendente.

Una panoramica della metodologia è presentato in Figura 2. Nel lavoro qui presentato, cellule CHO sono state transfettate con il vettore FIBS e linee cellulari stabili sono stati selezionati con una pressione antibiotico di 400 mcg / ml Zeocin. Due linee separate cellulari stabili esprimenti costitutivamente i sensori di glucosio e glutammina sono stati quindi creati. Figura 1 illustra le configurazioni delle due biosensori utilizzati in questo studio. Il sensore di glucosio si basa su...

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

FIBS-enabled Noninvasive Metabolic Profiling

Una descrizione di come calibrare trasferimento Förster Resonance Energy sensori biologici integrati (FIBS) per in situ profilo metabolico è presentato. Le FIBS possono essere utilizzati per misurare i livelli intracellulari di metaboliti non invasivo aiutando nello sviluppo di modelli metabolici e high throughput screening delle condizioni bioprocesso.

FIBS-enabled non invasiva Metabolic Profiling

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 Views.  Imperial College London.  Una descrizione di come calibrare trasferimento Förster Resonance Energy sensori biologici integrati (FIBS) per in situ profilo metabolico è presentato. Le FIBS possono essere utilizzati per misurare i livelli intracellulari di metaboliti non invasivo aiutando nello sviluppo di modelli metabolici e high throughput screening delle condizioni bioprocesso. 

Video: 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.

Alta MicroRNA profiling Throughput: Multiplex Ottimizzato qRT-PCR a scala nanoliter sul Fluidigm dinamica IFCS ArrayTM

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

Ricerca

Bioingegneria

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 carico di compressione e Correlativo imaging non invasiva del Bone-parodontale legamento-dente Fibrosa comune

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.

Quantificazione e Size-profiling dei extracellulare vescicole Utilizzando sintonizzabile resistivo 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.

Facile e preciso meccano-profiling su micropost Array

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.

Glycoengineering metabolica di acido sialico utilizzando N-acil-per volta 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.

Supporto metabolico di asportato, tessuti di cervello viventi durante l'acquisizione di microscopia a risonanza magnetica

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: Un approccio potente, non invasivo per studiare il metabolismo energetico cellulare

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.

Nanosensori per rilevare attività della proteasi In Vivo per la diagnostica non invasiva

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.

Misurazione della proteinasi metallica della matrice cellulare globale e l'attività metabolica in idrogel 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 modello di coltura a matrice extracellulare extracellulare -adipocyte umano per lo studio del crosstalk metabolico a matrice

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