Name: quantitative analysis of random migration of cells using time-lapse video microscopy
Uploaded: 2012-05-13T14:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

Forfatterne vil gerne takke Amanda Struckhoff for den indledende hjælp til eksperimentet. Dette arbejde blev støttet af en bevilling fra NIH 5RO1CA115706.

1. Udarbejdelse af plade med Collagen <ol><li> Fortyndes collagen til en slutkoncentration på 10 ug / ml i Opti-MEM-medium. </li><li> Coat 6-brøndsplader med collagen, ved tilsætning af 1,5 ml fra trin 1 til hver brønd. Inkuber natten over ved 4 ° C. </li><li> Den følgende dag vaskes pladen 2 gange med 1X phosphatpufret saltvand (PBS) og lagrer dem i PBS. </li></ol> 2. Cellepræparat og podning på en 6-brønds plade Bemærk: Brug varme medier o...

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84, 359-369 (1996).</li><li>Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+integrating+signals+from+front+to+back.">Cell migration: integrating signals from front to back.</a> Science. 302, 1704-1709 (2003).</li><li>Petrie, R. J., Doyle, A. D., 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=Random+versus+directionally+persistent+cell+migration.">Random versus directionally persistent cell migration.</a> Nat. Rev. Mol. Cell Biol. 10, 538-549 (2009).</li><li>Valone, F. H., Austen, K. F., Goetzl, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+the+random+migration+of+human+platelets.">Modulation of the random migration of human platelets.</a> J. Clin. Invest. 54, 1100-1106 (1974).</li><li>Biehlmaier, O., Hehl, J., Csucs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+speed+comparison+of+microscope+software+programs.">Acquisition speed comparison of microscope software programs.</a> Microsc. Res. Tech. 74, 539-545 (2011).</li><li>Struckhoff, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+regulation+of+ROCK+in+tumor+cells+controls+CXCR4-driven+adhesion+events.">Dynamic regulation of ROCK in tumor cells controls CXCR4-driven adhesion events.</a> J. Cell. Sci. 123, 401-412 (2010).</li><li>Abramoff, M. D., Magelhaes, P. J., Ram, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophoton. Internat. 11, 36-42 (2004).</li><li>Greenberg, J. H., Seppa, S., Seppa, H., Tyl Hewitt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+collagen+and+fibronectin+in+neural+crest+cell+adhesion+and+migration.">Role of collagen and fibronectin in neural crest cell adhesion and migration.</a> Dev. Biol. 87, 259-266 (1981).</li><li>Albini, A., Sporn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumour+microenvironment+as+a+target+for+chemoprevention.">The tumour microenvironment as a target for chemoprevention.</a> Nat. Rev. Cancer. 7, 139-147 (2007).</li><li>Yu, Y. P., Luo, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopodin-mediated+suppression+of+prostate+cancer+cell+migration+involves+interaction+with+zyxin.">Myopodin-mediated suppression of prostate cancer cell migration involves interaction with zyxin.</a> Cancer. Res. 66, 7414-7419 (2006).</li><li>Teng, T. S., Lin, B., Manser, E., Ng, D. C., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+promotes+directional+cell+migration+by+regulating+Rac1+activity+via+its+activator+betaPIX.">Stat3 promotes directional cell migration by regulating Rac1 activity via its activator betaPIX.</a> J. Cell. Sci. 122, 4150-4159 (2009).</li><li>Wozniak, M. A., Kwong, L., Chodniewicz, D., Klemke, R. L., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R-Ras+controls+membrane+protrusion+and+cell+migration+through+the+spatial+regulation+of+Rac+and+Rho.">R-Ras controls membrane protrusion and cell migration through the spatial regulation of Rac and Rho.</a> Mol. Biol. Cell. 16, 84-96 (2005).</li><li>Orr, A. W., Elzie, C. A., Kucik, D. F., Murphy-Ullrich, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondin+signaling+through+the+calreticulin/LDL+receptor-related+protein+co-complex+stimulates+random+and+directed+cell+migration.">Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration.</a> J. Cell Sci. 116, 2917-2927 (2003).</li><li>Smith, A., Bracke, M., Leitinger, B., Porter, J. C., Hogg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LFA-1-induced+T+cell+migration+on+ICAM-1+involves+regulation+of+MLCK-mediated+attachment+and+ROCK-dependent+detachment.">LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment.</a> J. Cell Sci. 116, 3123-3133 (2003).</li></ol>

Den virkelige tid tilfældige migration analysen muliggør præcis, følsom, analyse af cellemigration parametre såsom hastighed og forskydning. Denne fremgangsmåde er ikke begrænset til slutpunkt måleværdien, og det er derfor mere kvantitativ. Da cellerne overvåges under normal DMEM-medium med serum, og det reducerer den skadelige virkning af længere inkubation i serumfrie medier. Det har vist sig, at migreringen af celler afhænger af formateringen og bryde celle-kontakter med substratet 8, således ...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> DMEM </td><td> Thermo Scientific </td><td> SH30243.01 </td><td></td></tr><tr><td> FBS </td><td> Gemini bioprodukter </td><td> 100-106 </td><td></td></tr><tr><td> Opti-Mem </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Collagen </td><td> BD </td><td> 354231 </td><td></td></tr><tr><td> Mikroskop med Live Cell kammer &amp; Automated XY-scene controller </td><td> Olympus </td><td> Olympus 1X81 </td><td></td></tr><tr><td> Miljøkontrol kammer </td><td> Neue </td><td> Neue Live-Cell Afdeling </td><td> Vores har en specialbygget rektangulær glasplade top til passende optik gennem toppen af ​​kammeret. Den rektangulære form plads multi-brønds plader, der anvendes i mange af vores eksperimentellets. </td></tr><tr><td> Automatiseret XY-scene </td><td> Forud </td><td> Forud ProScan </td><td> Dette muliggør præcis tilbagevenden til formere udvalgte XY positioner under tidsforløbet mikroskopi. </td></tr><tr><td> Kamera </td><td></td><td> Hamamatsu EM kamera C9100 </td><td></td></tr><tr><td> Software </td><td> Typisk købes via mikroskop leverandør såsom Olympus </td><td> Slide Book 5 </td><td></td></tr></tbody></table>

quantitative analysis of random migration of cells using time-lapse video microscopy

Cellemigration er en dynamisk proces, hvilket er vigtigt for embryonisk udvikling, vævsreparation, immunsystem, og tumorinvasion 1, 2. Under retningsbestemt migration, bevæger celler hurtigt i respons på et ekstracellulært kemotaktisk signal, eller som reaktion på iboende signaler 3, som det grundlæggende motilitet maskinen. Tilfældigt migration forekommer, når en celle har lav indre retningsbestemthed, således at cellerne undersøge deres lokale miljø. Cell migration er en kompleks proces, i det første svar cellen undergår polarisering og udvider fremspring i retning af migration 2. Traditionelle metoder til måling af migration såsom Boyden-kammer migration assay er en let metode til at måle kemotaksi in vitro, hvilket muliggør måling migration som et slutpunkt resultat. Men denne fremgangsmåde hverken tillader måling af de enkelte migration parametre, ligesom det heller ikke muligt at visuasering af morfologiske ændringer, som cellen undergår i løbet af migration. Her præsenterer vi en metode, der giver os mulighed for at overvåge vandrende celler i realtid ved hjælp af video - tidsforskydningen mikroskopi. Siden celle migration og invasion er kendetegnende for kræft, vil denne metode kunne anvendes til at studere kræft celle migration og invasion in vitro. Tilfældig migration af blodplader er blevet betragtet som en af parametrene for trombocytfunktionen 4, derfor denne metode kunne også være nyttigt i at studere blodplade funktioner. Dette assay har den fordel at være hurtig, pålidelig, reproducerbar og ikke kræver optimering af celleantal. For at opretholde fysiologisk egnede betingelser for celler, er mikroskop udstyret med CO2 forsyning og temperatur termostat. Cellebevægelse overvåges ved at tage billeder ved hjælp af et kamera er monteret på mikroskop ved regelmæssige mellemrum. Cellemigration kan beregnes ved at måle den gennemsnitlige hastighed, og enGennemsnitligt forskydning, der beregnes ved Slidebook software.

Watch this Scientific Journal Video about Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy at JoVE.com

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Denne fremgangsmåde tillader overvågning af celler i realtid og kvantitative målinger af forskellige cellemigrering parametre såsom hastighed, forskydning og hastighed. I modsætning til de traditionelle metoder, er dette realtime strategi ikke er baseret på endpoint kvantitative migration målinger, i stedet giver mulighed for overvågning og beregning af forskellige parametre kontinuerligt.

Kvantitativ analyse af Random migration af celler Brug af Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Research

JoVE Journal

Biology

Monitoring Actin Disassembly with Time-lapse Microscopy

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Brug af Time Lapse Microscopy at Visualiser Anoxia-induceret Suspended Animation i<em&gt; C. elegans</em&gt; Embryoner

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Kvantitativ analyse af Autophagy hjælp Advanced 3D fluorescensmikroskopi

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Visualisering af kraniofaciale udvikling i sox10: Kaede Transgene zebrafisk linje ved hjælp af Time-lapse konfokal mikroskopi

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-Lapse Video mikroskopi for Vurdering af EYFP-Parkin Aggregering som markør for Cellulær Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

Høj opløsning Time-lapse Imaging og Automatiseret analyse af mikrotubulusdynamik i levende menneske navlestrengsveneendotelceller

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Linse-gratis Video mikroskopi for dynamiske og kvantitativ analyse af vedhængende cellekultur

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Specifik mærkning af mitokondrielle nukleoider til tidsforfald struktureret belysning mikroskopi

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...