Name: creating adhesive and soluble gradients for imaging cell migration with fluorescence microscopy
Uploaded: 2013-04-04T13:00:00
Description: Watch this Scientific Journal Video about Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy at JoVE.com

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 pulizi...

<ol>
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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

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

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)

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

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

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

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

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

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 ...