Name: live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix
Uploaded: 2011-12-22T13:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

Vi takker Dr. Grant Sumida til kritisk læsning af manuskriptet. Dette arbejde blev støttet af en Beckman Young Investigator Award (SY), en Hellman Familie nyt fakultet Award (SY), en NIH EUREKA, University of California Cancer Research koordineringsudvalg.

1. Generering af stabile cellelinje (f.eks MDCK celler) <ol><li> Plate celler på 80-90% confluency i en P35 fad. Lad ikke celler danner 100% sammenflydende monolag, hvilket vil reducere transfektion effektivitet. </li><li> Transfect cellerne med plasmidet af interesse ved hjælp af Lipofectamine 2000. Optimer transfektion betingelser ved hjælp af producentens protokol. </li><li> Næste dag, passage cellerne i to P150 petriskåle. Det store fad anbefales at give tilstrækkelig afstand mellem den stabile kolonier. </l...

<ol>
	<li>Shih, W., Yamada, S., Myosin, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dependent+retrograde+flow+drives+3D+cell+migration.">A dependent retrograde flow drives 3D cell migration.</a> Biophys. J. 98 (8), L29-L31 (2010).</li><li>O'Brien, L. E., Yu, W., Tang, K., Jou, T. S., Zegers, M. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+biochemical+analysis+of+Rac1+in+three-dimensional+epithelial+cell+cultures.">Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.</a> Methods Enzymol. 406, 676-691 (2006).</li><li>Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tractions+exerted+by+cells+in+three-dimensional+matrices.">Measurement of mechanical tractions exerted by cells in three-dimensional matrices.</a> Nat. Methods. 7 (12), 969-971 (2010).</li><li>Del Alamo, J. C., Meili, R., Alonso-Latorre, B., Rodriguez-Rodriguez, J., Aliseda, A., Firtel, R. A., Lasheras, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+analysis+of+eurkaryotic+cell+motility+by+improved+force+cytometry.">Spatio-temporal analysis of eurkaryotic cell motility by improved force cytometry.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (33), 13343-13348 (2007).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nat. Rev. Mol. Cell Biol. 2 (6), 444-456 (2001).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modeling+approaches+to+in+vivo+imaging.">Kinetic modeling approaches to in vivo imaging.</a> Nat. Rev. Mol. Cell Biol. 2 (12), 898-907 (2001).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+toolbox+for+assessing+protein+location+and+function.">Fluorescent toolbox for assessing protein location and function.</a> Science. 312 (5771), 217-224 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2, 905-909 (2005).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li></ol>

Her beskriver vi en metode til at bruge live-cell imaging at studere de mekanismer af celle migration i en tre-dimensionel matrix. Succesen med denne teknik er afhængig af at opnå &quot;god&quot; kloner stabilt udtrykker GFP-taggede proteiner. Det lave niveau for GFP proteiner vil kræve et overskud excitation eksponering, der kompromiser celle sundhed, mens for højt GFP niveau vil have uønskede bivirkninger på cellen. Således vektoren valg til transfect gener i celler er vigtig (f.eks initiativtageren, fluorescen...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Kollagen, kvæg, Type I </td><td> BD Biosciences </td><td> 354231 </td><td> Stock er omkring 3 mg / ml </td></tr><tr><td> 3-aminopropyltrimethoxysilane </td><td> Sigma Aldrich </td><td> 281778 </td><td> Fortyndes i vand </td></tr><tr><td> glutaraldehyd </td><td> Sigma Aldrich </td><td> 340855 </td><td> Fortyndes i PBS </td></tr><tr><td> 1M HEPES </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> Fluospheres polystyren mikrokugler 1 μm, rød fluorescens (580/605) </td><td> Invitrogen </td><td> F13083 </td><td></td></tr><tr><td> Geneticin (G418) </td><td> Invitrogen </td><td> 11811-031 </td><td></td></tr&gt; <tr><td colspan="4"> Dyrkningsmedier komponenter: </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31600-034 </td><td></td></tr><tr><td> Føtalt bovint serum </td><td> Atlanta Biologicals </td><td> S115500 </td><td></td></tr><tr><td> Penicillin / Streptomycin </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Kanamycin </td><td> Invitrogen </td><td> 15160-054 </td><td></td></tr></tbody></table>

live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix

Traditionelt har cellemigration blevet undersøgt på to-dimensionelle, stiv plast overflader. Men i vigtige biologiske processer som sårheling, vævsregeneration, og kræft metastase, må cellerne navigere gennem komplekse, tre-dimensionelle ekstracellulære væv. For bedre at forstå mekanismerne bag disse biologiske processer, er det vigtigt at undersøge rollerne for de proteiner, der er ansvarlig for kørsel celle migration. Her skitserer vi en protokol til at studere de mekanismer for celle migration ved hjælp af epitelcelle linje (MDCK), og en tre-dimensionel, fiberholdigt, self-polymerisering matrix som model system. Denne optisk klar ekstracellulære matrix er let modtagelig for live-cell imaging studier og bedre efterligner den fysiologiske, blødt væv miljø. Denne rapport viser en teknik til direkte at visualisere protein lokalisering og dynamik, og deformation af det omgivende tredimensionale matrix. Eksamennering af protein lokalisering og dynamik i løbet af cellulære processer giver nøglen indsigt i protein funktioner. Genetisk kodet fluorescerende tags give en unik metode til at observere protein lokalisering og dynamik. Ved hjælp af denne teknik, kan vi analysere subcellulære ophobning af centrale, kraft-generering cytoskeletal komponenter i real-tid som cellen manøvrer gennem matrix. Hertil kommer, bruger flere fluorescerende tags med forskellige bølgelængder kan vi undersøge lokalisering af flere proteiner samtidigt, hvilket giver os mulighed for at teste, for eksempel om forskellige proteiner har lignende eller divergerende roller. Desuden kan dynamikken i fluorescens-mærkede proteiner skal kvantificeres med fluorescerende Recovery Efter fotoblegning (FRAP) analyse. Denne måling assays proteinet mobilitet, og hvordan stabilt bundet til proteiner er til cytoskeletal netværket. Ved at kombinere live-cell imaging med behandlingen af ​​protein funktion hæmmere,vi kan undersøge i real-time ændringer i fordelingen af ​​proteiner og morfologi af trækkende celler. Desuden har vi også kombinere live-cell imaging med brug af fluorescerende sporstof partikler indlejret i den matrix, for at visualisere den matrix deformation celle migration. Således kan vi visualisere, hvordan en vandrende celle distribuerer kraft-genererende proteiner, og hvor trækkraft er, at kræfter til det omgivende matrix. Gennem disse teknikker, kan vi få værdifuld indsigt i de roller af specifikke proteiner og deres bidrag til de mekanismer af celle migration.

Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Cellulære processer såsom celle migration er traditionelt blevet undersøgt på to-dimensionelle, stiv plast overflader. Denne rapport beskriver en teknik til direkte at visualisere protein lokalisering og analysere protein dynamik i celler vandrer ud i en mere fysiologisk relevante, tre-dimensionel matrix.

Live-cell imaging at migrere celler, der udtrykker fluorescens-mærkede Proteiner i en tre-dimensionel Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Planar and Three-Dimensional Printing of Conductive Inks

Planar-og tredimensionelle Udskrivning af ledende blæk

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

Universal Håndholdt Tre-dimensionel Optoacoustic Imaging Probe for Deep Tissue Menneskelig angiografi og Funktionelle Prækliniske studier i Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Analyse af Cell Migration inden for en tre-dimensionel kollagenmatrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Brug af Cell-substrat Impedans og levende celler Imaging at måle realtid Ændringer i cellulær adhæsion og De-vedhæftning induceret af Matrix Ændring

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generation af en tre-dimensionel fuld tykkelse Skin Equivalent og Automated Anskydning

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Live Cell Imaging during Mechanical Stretch

Live-Cell Imaging under Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

Engineering Tredimensionale epitelvæv indlejret i ekstracellulær matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Smelte Electrospinning skrivning af tre-dimensionelle Poly(ε-caprolactone) stilladser med kontrollerbare morfologier for Tissue Engineering applikationer

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Dyrkning pattedyrsceller i tre-dimensionelle peptid stilladser

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...