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

Forfatterne erkjenner støtte fra den australske Forskningsrådet og National Health and Medical Research Council of Australia og liker også å takke Australian National Fabrication Facility for SU-8 master for microcontact utskrift. SHN støttes av departementet for høyere utdanning Malaysia og Universiti Sains Malaysia.

1. Passivisering av Glass Dekkglass med PLL-PEG-biotin Dette trinnet er utformet for å passivere overflaten slik at cellene fester seg og migrere bort bestemte områder på dekkglass som opprettes med microcontract trykkteknikker (Trinn 2-3a) eller dip penn litografi (Trinn 3b). For passivering, er en co-polymer av poly-L-lysin (PLL) og polyetylenglykol (PEG) som brukes i hvilken 20% av PEG molekyler blir podet til biotin (PLL-PEG-biotin). <ol><li> Å rengjøre glass dekkglass (18 mm x 18...

<ol>
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(22), e1065 (2008).</li><li>Shin, Y., 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=Microfluidic+assay+for+simultaneous+culture+of+multiple+cell+types+on+surfaces+or+within+hydrogels.">Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.</a> Nat. Protocols. 7, 1247-1259 (2012).</li><li>Qin, D., Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+lithography+for+micro-+and+nanoscale+patterning.">Soft lithography for micro- and nanoscale patterning.</a> Nat. Protocols. 5, 491-502 (2010).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. 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I denne protokollen, brukte vi en kommersielt tilgjengelig microfluidic enhet (klebrig-Slide chemotaxis 3D fra Ibidi) for å studere effektene av kjemisk modifiserte overflater og kjemotiltrekkende graderinger på celle migrasjon. Dette microfluidic oppsettet krever ikke flyt fordi gradienten er etablert ved diffusjon langs lengden av kanalen. Dette er viktig fordi strømning kunne differensielt påvirke langsomt migrerende celler slik som fibroblaster som danner stabile fokale adhesjoner og raske migrerende ...

Rettet celle migrasjon er en grunnleggende egenskap av mange celler og er en viktig del av mange normale fysiologiske prosesser, herunder embryoutvikling, forsvar mot infeksjoner og sårheling. I tillegg spiller cellemigrering også en fremtredende rolle i mange sykdommer som vaskulær sykdom, tumorcelle metastasering og kronisk inflammasjon 1,2. Mens de klassiske statene celle migrasjon - polarisering, protrusion forlengelse, dannelse av vedheft, kraft generasjon og bak tilbaketrekking - er generelt akseptert 3,4, har klarlegging av Spatiotemporal mekanismer som signal integrasjon er koordinert vært mer utfordrende. Den ekstracellulære matriks (ECM) fungerer som et substrat for celleadhesjon, og gjennom iboende kjemisk 5 og fysiske 6 mangfold, gir retningsbestemte signaler for celle navigering 7,8. I tillegg til disse lim pekepinner 9, oppløselige faktorer 10-12 &lt;/ Sup&gt; for eksempel chemokines og vekstfaktorer kan indusere rettet celle migrasjon gjennom kjemotiltrekkende reseptorer og deres nedstrøms motogenic signalveier. Foreløpig er det ikke kjent om engasjement og signalering gjennom adhesjonsreseptorer (dvs. integriner) eller kjemotiltrekkende reseptorer som reseptor tyrosinkinase (RTK) er dominerende. Det er heller ikke kjent om hierarkier av reseptorsystemer er celletype bestemt. Levende celle mikroskopi gir et vell av informasjon som ikke er tilgjengelig i bulk analyser og kan kombineres med microfluidic enheter å generere graderinger av immobilisert 13,14 og løselig pekepinner 15 16. Metoden beskrevet her anvender en serie enkle og etablerte trinn for overflatemodifisering sammen med et kommersielt tilgjengelig microfluidic kammer for å opprette en avbildning assay for celle migrasjon som lett kan gjennomføres på en cellebiologi laboratorium (figur 1). Den sammensattemicrofluidic enhet har optiske egenskaper som er sammenlignbare med glass 17 og gradienter av diffusjonsevne molekyler er stabil i minst 16 timer. Systemet kan brukes til epifluorescence og avanserte mikroskopi teknikker. I motsetning til andre chemotaxis oppsett 18, er dette systemet egnet for opptak langsomt migrere, adherente celler. Viktigere er systemet modulært og lett tillater innføring av alternative klebemiddel eller oppløselige migrasjon signaler og undersøkelse av forskjellige celletyper.

<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

Celler kan føle og migrere mot høyere konsentrasjoner av selvklebende signaler som de glykoproteiner av ekstracellulær matriks og løselige signaler som vekstfaktorer. Her, skissere vi en fremgangsmåte for å lage motstridende gradienter av klebende og løselig pekepinner i en microfluidic kammer, som er kompatibel med levende celle bildebehandling. En kopolymer av poly-L-lysin og polyetylenglykol (PLL-PEG) er anvendt for å passivere glass dekkglass og hindre ikke-spesifikk adsorpsjon av biomolekyler og celler. Neste, microcontact utskrift eller dip penn litografi er brukt til å lage sporene streptavidin på passiverte overflater for å tjene som forankringspunkter for biotinylert peptid arginin-glycin-asparaginsyre (RGD) som limet stikkordet. En microfluidic enheten er plassert på den modifiserte overflaten og brukes til å lage gradient av selvklebende pekepinner (100% RGD til 0% RGD) på streptavidin spor. Endelig, er det samme microfluidic enhet som brukes til å opprette en gradient av en kjemotiltrekkende sukh som føtalt bovint serum (FBS), som det løselige stikkordet i motsatt retning av gradienten av selvklebende signaler.

For å forstå hvordan celle integrere trekkende signaler 25, har vi utviklet en metode for å bildet celler med fluorescens mikroskopi som vandrer i et miljø med konkurrerende lim og løselig gradienter (figur 1). Selvklebende spor som inneholdt fluorescerende streptavidin og biotinylert RGD ble opprettet med microcontact trykte spor og dip penn litografi (figur 2). Vellykket microcontact trykkteknikker indikeres av linje profilen av fluorescensintensiteten tvers sporene de...

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

Fremgangsmåte for sammenstilling av klebende og løselig gradienter i en mikroskopi kammer for live celle migrasjon studier er beskrevet. De utviklet miljø kombinerer bunnstoff overflater og lim spor med løsning gradienter og derfor gjør det mulig å bestemme den relative betydningen av veiledning signaler.

Opprette Lim og Løselig overgangar for Imaging Cell Migration med fluorescens mikroskopi

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

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

Høyoppløselig Spatiotemporal Analyse av Receptor Dynamics av ​​Single-molekyl fluorescens mikroskopi

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

Fra Fast Fluorescens Imaging til Molecular Diffusion Lov om Aktive cellemembraner i et kommersielt mikroskop

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

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

Fluorescens-slukking av en Liposomalinnkapslet Nær-infrarød fluoroforen som et verktøy for<em&gt; I Vivo</em&gt; Optisk 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

Photoactivated Lokalisering Mikros med bimolecular fluorescens Komplemente (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

Polydimethylsiloxane-polykarbonat microfluidic Enheter for celle migrasjon Studies Under Perpendicular Kjemisk og Oksygen Graderinger

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

Gjennomføre flere Imaging modi med en fluorescens mikroskopet

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

Visualisere vedheft formasjon i celler ved hjelp av avansert spinner disken totalt interne refleksjon fluorescens mikroskopi

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

3D-analyse av multi-Cellular Svar å Chemoattractant graderinger

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