Gli autori riconoscono il finanziamento della Australian Research Council e National Health and Medical Research Council of Australia e anche ringraziare la Australian National impianto di produzione per il SU-8 master per la stampa microcontatto. SHN è sostenuta dal Ministero dell&#39;Istruzione Superiore e Malesia Universiti Sains Malaysia.

1. Passivazione di vetrini con PLL-PEG-biotina Questa fase ha lo scopo di passivazione della superficie in modo che le cellule aderiscono e migrare su regioni specifiche sul vetrino di vetro che vengono creati con la stampa microcontract (Fase 2-3a) o litografia dip penna (Step 3b). Per la passivazione, un co-polimero di poli-L-lisina (PLL) e polietilene glicole (PEG) viene utilizzato in cui il 20% delle molecole di PEG sono stati innestati biotina (PLL-PEG-biotina). <ol><li> Per la pulizia coprioggetto di vetro (18 mm x 18 mm), immergere in un rack in 100% di etanolo e la cremagliera in un bagno sonicating per 30 minuti. Asciugare i vetrini in aria o in corrente di azoto. </li><li> Successivamente, i vetrini sonicazione per 30 min in 1 M NaOH. Successivamente sciacquare le superfici in vetro con cautela immergendo il rack in un bicchiere pieno di 1.5 L Milli-Q H 2 O. Ripetere il processo di risciacquo tre volte utilizzando fresco Milli-Q H 2 O di volta in volta. Asciugare i vetrini inun forno a 60 ° C. </li><li> Mezzo posto del vetro pulito coprioggetti singolarmente in una camera umida, che è fatto di una piastra a 24 pozzetti parzialmente riempito con H 2 O. Goccia 15 ml di PLL biotina-PEG-(1 mg / ml) in PBS su ciascun vetrino coprioggetto. Prendere l&#39;altra metà dei restanti coprioggetto di vetro pulito e con attenzione a sandwich la soluzione PEG. Lasciare i vetrini per 1 ora nella camera umida. Far scorrere i coprioggetti sandwich delicatamente distanza l&#39;uno dall&#39;altro, senza raschiare il PEG superficie verniciata e sciacquarle con Milli-Q H 2 O. L&#39;acqua deve scorrere via facilmente sul lato PEG-trattati. Se necessario, asciugare i vetrini su carta assorbente con la superficie trattata rivolta verso l&#39;alto. Conservare i vetrini in un essiccatore sotto vuoto fino all&#39;uso successivo. </li></ol> 2. Fabbricazione di francobolli per la stampa microcontatto Descriviamo due processi alle tracce streptavidina modello sui vetrini passivati. Il primo, microfonorocontact stampa, tipicamente richiede la realizzazione di un master di silicio da cui viene colato un timbro PDMS. Nel nostro caso, il modello del timbro PDMS sono 100 um livello linee che sono distanziati 290 micron da centro a centro con un&#39;altezza caratteristica di 25 micron. Qui di seguito, si descrive brevemente come fabbricare un maestro adatto al silicio (Step 2,1-2,3) e timbro PDMS (Fase 2.4). Tuttavia, ci sono più protocolli per la stampa microcontatto descritti in letteratura 19-23, che sono adatti per la stampa su modelli proteici coprioggetto. Master può anche essere fatta su misura e acquistati da fonti commerciali. <ol><li> Prima, pulire il wafer di silicio (4 &quot;di diametro) incubando in soluzione Piranha (3:1 v: v di acido solforico e perossido di idrogeno al 30%). Per 20 min Sciacquare accuratamente il wafer 10 volte con MilliQ H 2 O. Essiccare il wafer in un forno a 150 ° CO / N. </li><li> Risciacquare il wafer di silicio pulita ed essiccata con il 100% di acetone, seguito da 100% di alcol isopropilico. Prossimospin-coat 2 ml di GM1070 SU-8 photoresist (Gersteltec Sarl) sul wafer a 3.100 rpm per 40 secondi per creare un 25 spesso strato micron. Il campione viene quindi cotta su una piastra calda a 65 ° C per 5 minuti seguiti da un ulteriore 5 min a 95 ° C. </li><li> Esporre il SU-8 fotosensibile ai raggi UV per 60 secondi sotto un fotomaschera con il modello desiderato (ad esempio utilizzando un Quintel Q6000 maschera allineatore) e ripetere il processo di cottura descritto al punto 2.2. Allora il campione è sviluppato immergendo il wafer in SU-8 developer (Gersteltec Sarl) per 4 min, sciacquati in 100% alcool isopropilico ed essiccato sotto flusso di azoto. L&#39;area del SU-8 photoresist che non è stato esposto a luce UV viene quindi rimosso, creando il pattern inverso del timbro PDMS. </li><li> Il passo successivo è quello di lanciare un timbro PDMS dal master silicio. Primo, mescolare 1 parte (w / w) del catalizzatore con 10 parti di prepolimero elastomero (Sylgard 184). Per eliminare eventuali bolle d&#39;aria, la miscela degas in un essiccatore collegato a una linea di vuotoper 1 ora. Successivamente, versare l&#39;elastomero PDMS sul master silicio all&#39;interno una capsula di Petri a circa 1 cm di altezza e curare la miscela master e PDMS in un forno a 70 ° C per 1 ora. Infine, il timbro PDMS viene rimosso dal master silicio e tagliato a misura. </li></ol> 3. Patterning streptavidina o Cue adesivi sul coprioggetto di vetro passivato Dopo passivante vetrini, profili proteici sono stampate con stampa microcontact (3a Fase) o dip-pen litografia (Step 3b). Nel nostro caso, streptavidina viene stampato come linee, che costituiscono la base per gradienti RGD (Passaggio 4). La stessa procedura può essere utilizzata anche per la stampa di proteine ​​di matrice per creare modelli adesivi. Un metodo alternativo per la stampa microcontatto per il patterning di biomolecole su superfici è litografia dip penna. In litografia dip penna, un cantilever viene utilizzato per trasferire un piccolo volume di soluzione sulla superficie. Le dimensioni dei cantilever, viscosità della soluzione, il tempo di contatto del cantilever sulla superficie, idrofobicità della superficie e l&#39;umidità nella camera di stampa determina la dimensione delle caratteristiche stampati. Il vantaggio di questo metodo è che diversi modelli possono essere stampati per ogni esperimento, in quanto non vi è alcuna necessità di fabbricare maestri e timbri. Lo svantaggio è che ci vuole più tempo per stampare un dato modello e che litografia dip penna necessita di uno strumento specializzato che potrebbe non essere disponibile in molti laboratori. La penna strumento tuffo litografia che abbiamo usato era l&#39;NanoeNabler da Bioforce nanoscienze. Qui, abbiamo stampato piccoli punti che erano &lt;10 micron di diametro, per il quale abbiamo usato SPT10 cantilever. 3a. Microcontact Stampa (μCP) Tecnica <ol><li> Per pulire e rendere il timbro PDMS più idrofila, trattare lato modellato dei francobolli PDMS in un UV e ozono (per esempio, Procleaner ozono UV Bioforce da Nanoscience) per 1,5 ore. In alternativa, utilizzare un plasmapulitore per un tempo più breve per lo stesso scopo. Questo processo crea una superficie ossidata PDMS 24, che migliora il processo di stampa. </li><li> Immediatamente dopo il trattamento con ozono o plasma, luogo e diffondere 10 microlitri di streptavidina (1 mg / ml in PBS) sui francobolli PDMS e lasciare per 1 ora in una camera umida (come parzialmente riempito piastra da 6 pozzetti, vedere il punto 1.3) . Se le tracce dovrebbero essere visibili al microscopio a fluorescenza, utilizzare streptavidina che è coniugato ad un fluorocromo come streptavidina-AlexaFluor350. </li><li> Rimuovere streptavidina in eccesso dal timbro con carta velina e asciugare all&#39;aria il timbro per circa 1 min. Premere leggermente il timbro sul PLL-PEG-biotina-rivestite coprioggetto di vetro. </li><li> Se streptavidina fluorescente è stata utilizzata in 3.a2 Passo, esaminare lo schema usando sotto un microscopio a epifluorescenza. Conservare le superfici stampate in un essiccatore. </li></ol> 3b. Dip Pen Litografia <ol><li> Primo, miscelare 1 parte di 1 mg / ml o streptavidina Streptavidin-AlexaFluor350 con 10 parti di glicerolo (v / v) e caricare 5 microlitri della miscela sulla cantilever. Poi inizia il processo di stampa come descritto nel manuale di istruzioni dello strumento litografia dip penna. Per ridurre la dimensione del punto di circa 5 micron, regolare la velocità di stampa, il tempo di contatto del cantilever sulla distanza o superficie verticale del cantilever alla superficie. Nell&#39;esempio illustrato in figura 2c, puntini sono stati stampati in una linea con riga e colonna di separazione impostato a 10-15 micron. Questa distanza è sufficiente per evitare punti di fusione in loro ma permette la visualizzazione di punti multipli in un unico campo di vista con impostazioni maggior microscopio. </li><li> Conservare le superfici stampate in un essiccatore. </li></ol> 4. Creazione di sfumature adesivo sul streptavidina-modello Superfici Creiamo gradienti adesive di biotina-RGD su vetrini rivestiti con streptavidina (Figura 3) o contain modelli streptavidina su vetrini passivato (Figura 5). Per creare un gradiente di superficie, è stato utilizzato un dispositivo disponibile in commercio microfluidica (chiamato sticky-Slide chemiotassi 3D Ibidi) che crea un gradiente stabile soluzione per diffusione passiva. Competere per i siti di legame streptavidina, abbiamo impiegato due opposti gradienti di biotina-RGD e biotina che non era coniugato RGD (vedi Figura 1). <ol><li> Rispettare le streptavidina rivestite coprioggetto di vetro o con motivi sul lato adesivo del appiccicoso-Slide dispositivo chemiotassi 3D. Per garantire che non vi siano perdite per diverse ore, i bordi del dispositivo sono sigillate con un sottile strato di vaselina riscaldato e con un secondo strato di una miscela di 1 parte di vaselina 1 parte di paraffina (w / w). </li><li> Riempire il canale del dispositivo con 6 microlitri PBS nel canale. Quindi riempire i due serbatoi con 70 microlitri di 0,03 mg / pl biotina-4-fluoresceina in PBS e 70 &amp; mu; l di 0,03 mg / pl biotina-RGD in PBS, rispettivamente. Incubare i campioni a temperatura ambiente al buio per 1 ora. </li><li> Rimuovere la biotina soluzioni e risciacquare accuratamente la superficie mentre è ancora collegata al dispositivo microfluidico due volte con PBS. Segnare la direzione del gradiente adesivo e, se necessario, lasciare il dispositivo riempito con PBS a 4 ° C per ma assicurare che il dispositivo non si asciughi. Se necessario, analizzare il gradiente adesivo con un microscopio a fluorescenza. </li></ol> 5. Etichettatura di celle con fluorofori per l&#39;imaging cellulare in diretta Le cellule sono etichettati con coloranti fluorescenti che aiutano il monitoraggio delle cellule sotto il microscopio a fluorescenza. Sonde comuni sono marcatori fluorescenti organelli, come Syto 64 Rosso, che etichetta il nucleo cellulare. Per fare ciò, le cellule vengono prima lavati tre volte con PBS e poi incubate con 1 pM Syto 64 Rosso in terreni di coltura per 45 min ed infine lavato altre tre volte con PBS. Si prega di notare che le cellule non deve esseremantenuto in PBS per lunghi periodi di tempo. Coloranti alternativi per il monitoraggio delle cellule sono serie CellTracker che macchia il citoplasma e vengono mantenuti durante la migrazione e la divisione cellulare. Trasfezione di cellule con proteina di fusione fluorescente è particolarmente utile per registrare la distribuzione subcellulare di una proteina di interesse durante la migrazione. Tuttavia, proteine ​​fluorescenti tipicamente foto-bleach veloce di coloranti organici così che osservazioni a lungo termine può non essere possibile. 6. Caricamento di celle con una sfumatura solubile nel dispositivo microfluidica <ol><li> Contare fluorescente cellule marcate e divise in due provette di numero di cellule uguali. Lavare e risospendere un tubo di cellule con supporti contenenti il ​​fattore chemiotattico, come il basso glucosio Eagle modificato da Dulbecco (DMEM) con 10% di siero bovino fetale (FBS). Lavare e risospendere l&#39;altro tubo di cellule nei media senza il chemiotattico, cioè DMEM con 0% FBS. La concentrazione finaledi cellule in ciascun tubo deve essere di 5 x 10 5 cellule / ml. </li><li> Rimuovere tutte le soluzioni dal dispositivo microfluidica (dal punto 4.3) e posizionarlo sul palco microscopio (vedi punto 7). Carico 70 microlitri di cellule (5 x 10 5 cellule / ml in DMEM 0% FBS) in un serbatoio e 70 microlitri di cellule HeLa (5 x 10 5 cellule / ml in DMEM 10% FBS) in un altro. Liberamente inserire un nastro sopra i serbatoi per evitare l&#39;evaporazione. Se le cellule sulla superficie sono pre-incubato prima imaging, è opportuno che le cellule vengono caricate nel dispositivo in terreni di coltura senza il gradiente. Il dispositivo viene quindi posto sul piatto microscopio e mezzi sostituito con gradiente supporti cioè DMEM senza FBS in un serbatoio e DMEM con 10% FBS nell&#39;altro. </li></ol> 7. Vivo imaging cellulare e analisi dei dati <ol><li> Accendere il microscopio (Nikon Ti-E) e riscaldare il palco, almeno 1 ora prima di avviare l&#39;esperimento. </li><li> Assicurarsi che il canale del micrDispositivo ofluidic è nel campo di vista e cellule sono a fuoco. Selezionare il parametro di acquisizione immagine, come i tempi di esposizione e filtri fluorescenti, sequenza di immagini del campo chiaro e fluorescenza in grado di catturare le cellule e le tracce così come i punti di tempo e la durata di registrazione (ad esempio ogni 15 minuti per 16 ore). </li><li> Analizzare i dati con i software appropriati quali ImageJ Allineamento manuale o Ibidi chemiotassi e strumento di migrazione. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanolithography+and+nanochemistry:+probe-related+patterning+techniques+and+chemical+modification+for+nanometer-sized+devices.">Nanolithography and nanochemistry: probe-related patterning techniques and chemical modification for nanometer-sized devices.</a> Angewandte Chemie (International ed. in English). 43, 2480-2495 (2004).</li><li>Xia, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directional+control+of+cell+motility+through+focal+adhesion+positioning+and+spatial+control+of+Rac+activation.">Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation.</a> FASEB J. 22, 1649-1659 (2008).</li><li>Oakes, P. W., Beckham, Y., Stricker, J., Gardel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tension+is+required+but+not+sufficient+for+focal+adhesion+maturation+without+a+stress+fiber+template.">Tension is required but not sufficient for focal adhesion maturation without a stress fiber template.</a> The Journal of Cell Biology. 196, 363-374 (2012).</li></ol>

Noi non abbiamo interessi commerciali da dichiarare.

In questo protocollo, abbiamo utilizzato un dispositivo disponibile in commercio microfluidica (sticky-Slide chemiotassi 3D Ibidi) per studiare gli effetti delle superfici chimicamente modificati e le sfumature chemoattractant sulla migrazione delle cellule. Questa configurazione microfluidica non richiede un flusso perché il gradiente è stabilito per diffusione lungo la lunghezza del canale. Questo è importante perché il flusso differenziale potrebbe influenzare le cellule migrano lentamente come i fibro...

Migrazione cellulare Regia è una proprietà fondamentale di molte cellule ed è un aspetto fondamentale di molti processi fisiologici, tra cui lo sviluppo embrionale, la difesa contro le infezioni e guarigione della ferita. Inoltre, la migrazione cellulare gioca anche un ruolo importante in molte malattie quali malattia vascolare, cellule tumorali e metastasi 1,2 infiammazione cronica. Mentre gli stati classici della migrazione cellulare - polarizzazione, estensione protrusione, la formazione di adesione, la generazione di forza e retrazione posteriore - sono generalmente accettate 3,4, la delucidazione dei meccanismi spazio-temporali da cui esso è coordinato integrazione del segnale è stato più impegnativo. La matrice extracellulare (ECM) serve come substrato per l&#39;adesione cellulare, e attraverso chimica e fisica inerente 5 6 diversità, fornisce indicazioni direzionali per cella 7,8 navigazione. In aggiunta a questi segnali adesive 9, fattori solubili 10-12 &lt;/ Sup&gt;, come chemochine e fattori di crescita in grado di indurre la migrazione delle cellule attraverso i recettori chemiotattico diretto e la loro segnalazione a valle motogenic. Attualmente, non è noto se l&#39;impegno e segnalamento attraverso recettori di adesione integrine (cioè) o recettori chemoattraente, come recettore tirosina chinasi (RTK), sono dominanti. Non è neppure noto se gerarchie di sistemi recettoriali sono tipo specifico delle cellule. Microscopia di cellule in diretta dà una ricchezza di informazioni che non è accessibile nei test di massa e può essere combinato con dispositivi microfluidici per generare gradienti immobilizzati di spunti 13,14 e solubile 15 16. Il metodo qui descritto utilizza una serie di semplici passaggi e consolidata per modificazione superficiale in combinazione con una camera disponibile in commercio microfluidica per creare un saggio di imaging per la migrazione cellulare che può essere facilmente implementato in un laboratorio di biologia cellulare (Figura 1). L&#39;assemblatodispositivo a microfluidi ha qualità ottiche che sono paragonabili al vetro 17 e il gradiente di molecole diffusibili sono stabili per almeno 16 ore. Il sistema può essere utilizzato per tecniche epifluorescenza e avanzate. A differenza di altre configurazioni chemiotassi 18, questo sistema è adatto per la registrazione lenta migrazione, cellule aderenti. Importante, il sistema è modulare e permette facilmente l&#39;introduzione di adesivo alternativa o segnali migrazione solubili e l&#39;esame di vari tipi cellulari.

<table border="1">
	<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Coverglass staining outfits</td>
 <td>Thomas Scientific</td>
 <td>8542 E40</td>
 <td>Coverslip rack</td>
 </tr>
 <tr>
 <td>Oven</td>
 <td>Binder</td>
 <td></td>
 <td>ED 53 series</td>
 </tr>
 <tr>
 <td>Silicon wafer</td>
 <td>Silicon Quest</td>
 <td>708-007</td>
 <td>Boron doped &lt;100&gt; wafer, 4" diameter, 500 &mu;m, single side polished</td>
 </tr>
 <tr>
 <td>GM1070 SU-8 photoresist</td>
 <td>Gersteltec Sarl</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SU-8 developer</td>
 <td>Gersteltec Sarl</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 curing agent</td>
 <td>Dow Corning</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Sylgard 184 elastomer prepolymer</td>
 <td>Dow Corning</td>
 <td></td>
 <td >&nbsp;</td>
 </tr>
 <tr>
 <td>PLL-PEG-biotin (20%)</td>
 <td>SuSos AG</td>
 <td>PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)</td>
 <td>1 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Fluorescein</td>
 <td>Sigma</td>
 <td>46955</td>
 <td>1 mM in PBS</td>
 </tr>
 <tr>
 <td>Streptavidin-AlexaFluor350</td>
 <td>Invitrogen</td>
 <td>S-11249</td>
 <td>1 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Biotin-4-fluorescein</td>
 <td>Invitrogen</td>
 <td>B-1370</td>
 <td>0.03 &mu;g/&mu;l in PBS</td>
 </tr>
 <tr>
 <td>Biotin-RGD</td>
 <td>GenScript</td>
 <td>SC1208</td>
 <td>0.03 mg/ml in PBS</td>
 </tr>
 <tr>
 <td>Syto 64 Red</td>
 <td>Invitrogen</td>
 <td> S-11346 </td>
 <td>1 &mu;M in PBS</td>
 </tr>
 <tr>
 <td>Sticky slide chemotaxis 3D</td>
 <td>Ibidi</td>
 <td>80328</td>
 <td></td>
 </tr>
 <tr>
 <td>200 &mu;l Greiner yellow bevelled tip</td>
 <td>Greiner Bio-One</td>
 <td>739261</td>
 <td></td>
 </tr>
 <tr>
 <td>Vaseline</td>
 <td>Sigma</td>
 <td>16415</td>
 <td></td>
 </tr>
 <tr>
 <td>Paraffin wax, mp 55-57 &deg;C</td>
 <td>Sigma</td>
 <td>327204</td>
 <td></td>
 </tr>
 <tr>
 <td>Nano eNabler 10 &mu;m cantilever </td>
 <td>BioForce</td>
 <td>SPT-S-C-10s</td>
 <td></td>
 </tr>
 <tr>
 <td>Image J software</td>
 <td>National Institute of Health</td>
 <td></td>
 <td ><a href="http://rsbweb.nih.gov/ij/download.html" target="_blank">rsbweb.nih.gov/ij/download.html</a></td>
 </tr>
 <tr>
 <td>Manual Tracking plugin</td>
 <td>Fabrice Cordeli&egrave;res</td>
 <td></td>
 <td ><a href="http://rsb.info.nih.gov/ij/plugins/track/track.html" target="_blank">rsb.info.nih.gov/ij/plugins/track/track.html</a></td>
 </tr>
 <tr>
 <td>Chemotaxis and Migration Tool</td>
 <td>Ibidi GmbH</td>
 <td></td>
 <td><a href="http://www.ibidi.com/applications/ap_chemotaxis.html" target="_blank">www.ibidi.com/applications/ap_chemotaxis.html</a></td>
 </tr>
	 </tbody>
 </table>

creating adhesive and soluble gradients for imaging cell migration with fluorescence microscopy

Cellule possono percepire e migrare verso concentrazioni più elevate di stimoli adesive come le glicoproteine ​​della matrice extracellulare e spunti solubili come fattori di crescita. Qui, descriviamo un metodo per creare gradienti opposti di segnali adesivi e solubile in una camera microfluidica, che è compatibile con imaging cellulare vivo. Un copolimero di poli-L-lisina e polietilene glicole (PEG-PLL) è impiegato per passivare coprioggetto e prevenire adsorbimento non specifico di biomolecole e cellule. Successivamente, la stampa microcontatto o litografia dip penna sono utilizzati per creare percorsi di streptavidina sulle superfici passivati ​​per servire come punti di ancoraggio per il peptide biotinilato arginina-glicina-acido aspartico (RGD) come cue adesivo. Un dispositivo microfluidico è disposto sulla superficie modificata e utilizzata per creare il gradiente di segnali adesivi (100% allo 0% RGD RGD) sulle tracce streptavidina. Infine, lo stesso dispositivo microfluidico è usato per creare un gradiente di suc chemoattractanth come siero bovino fetale (FBS), come cue solubile in direzione opposta del gradiente di segnali adesivi.

Per capire come integrare i segnali di cellule migratorie 25, abbiamo sviluppato un metodo per celle di immagine con microscopia a fluorescenza che migrano in un ambiente con concorrenti gradienti adesivi e solubili (Figura 1). Tracce adesive che contenevano streptavidina fluorescente e RGD biotinilato sono stati creati con le tracce microcontatto stampati e litografia dip penna (Figura 2). Stampa microcontact successo è indicato dalla linea profilo dell&#39;intensità di fl...

Watch this Scientific Journal Video about Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy at JoVE.com

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Un metodo per l&#39;assemblaggio di gradienti adesivi e solubile in una camera per gli studi di microscopia vivi migrazione cellulare è descritto. L&#39;ambiente di ingegneria combina superfici antivegetative e le tracce adesive con pendenze soluzione e permette quindi di determinare l&#39;importanza relativa di spunti di orientamento.

Creazione di sfumature adesivi e solubile per la migrazione di imaging cellulare con microscopia a fluorescenza

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

creating-adhesive-soluble-gradients-for-imaging-cell-migration-with

Research

JoVE Journal

Bioengineering

12.5K Views.  The University of New South Wales. Un metodo per l'assemblaggio di gradienti adesivi e solubile in una camera per gli studi di microscopia vivi migrazione cellulare è descritto. L'ambiente di ingegneria combina superfici antivegetative e le tracce adesive con pendenze soluzione e permette quindi di determinare l'importanza relativa di spunti di orientamento.

Video: Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (&gt;3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Inserimento di Flexible Neural Sonde Uso Rinforzi rigida collegata con Biodissolvable adesivo

Microelectrode  arrays for neural interface devices that are made of biocompatible thin-film  polymer are expected to have extended functional lifetime ...

Ricerca

Bioingegneria

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Ad alta risoluzione Spatiotemporal Analisi del recettore Dynamics per singola molecola di microscopia a fluorescenza

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

Da Veloce imaging di fluorescenza di Molecular legge Diffusione su membrane cellulare dal vivo in un microscopio commerciale

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Fluorescenza tempra di un vicino infrarosso Fluoroforo incapsulata all&#39;interno di liposomi come strumento per<em&gt; In Vivo</em&gt; Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Fotoattivato localizzazione Microscopia con complementazione fluorescenza biomolecolare (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

Polidimetilsilossano-policarbonato dispositivi microfluidici per Cell Migration Studies Sotto perpendicolare chimica e gradienti di ossigeno

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Lo svolgimento di diverse modalità di Imaging con un microscopio a fluorescenza

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Visualizzazione di formazione di aderenze in cellule per mezzo di avanzati filatura microscopia di fluorescenza di riflessione interna totale disco

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Analisi 3D delle risposte multi-cellulari ai gradienti chemoattractant

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Microscopia di trazione integrata con microfluidica per la migrazione collettiva chemiotattica

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...